Attaching a stem-loop oligonucleotide to a double stranded DNA molecule

ABSTRACT

The present invention concerns preparation of DNA molecules, such as a library, using a stem-loop oligonucleotide. In particular embodiments, the invention employs a single reaction mixture and conditions. In particular, at least part of the inverted palindrome is removed during the preparation of the molecules to facilitate amplification of the molecules. Thus, in specific embodiments, the DNA molecules are suitable for amplification and are not hindered by the presence of the palindrome.

The present application is a continuation of U.S. application Ser. No.13/286,937 filed Nov. 1, 2011, now U.S. Pat. No.8,399,199, which is acontinuation of U.S. application Ser. No. 12/892,359 filed Sep. 28,2010, now U.S. Pat. No. 8,071,312, which is a continuation of U.S.application Ser. No. 12/270,850 filed Nov. 13, 2008, now U.S. Pat. No.7,803,550, which is a continuation of U.S. patent application Ser. No.11/366,222, filed Mar. 2, 2006, now abandoned, which claims priority toU.S. Provisional Patent Application Ser. No. 60/704,932, filed Aug. 2,2005, are incorporated by reference herein in their entirety.

FIELD OF THE INVENTION

The present invention generally concerns the fields of molecular biologyand cellular biology. In particular, the present invention regardspreparation and amplification of molecules, optionally utilizing a novelsingle-step reaction.

BACKGROUND OF THE INVENTION

Enzymatic reactions that involve DNA and RNA are numerous, and they playa central role in maintenance and propagation of living cells. Since thediscovery of the DNA double helix structure in 1953, researchers havefound, isolated and introduced into practice a multitude of differentenzymes that can, for example, cut, nick, trim, join, unwind,phosphorylate, de-phosphorylate, methylate, de-methylate, recombine,replicate, transcribe, repair, and perform many other reactions withnucleic acid molecules. These enzymes are now actively used in manyareas of biology, biotechnology and medicine to clone, amplify,sequence, identify mutations, quantify gene copy number, establishexpression patterns, determine DNA methylation status, etc.

Frequently the process of DNA analysis involves multiple enzymaticreactions that are performed in a sequential manner, with intermediatepurification steps between the reactions. Sometimes, the reactions aremultiplexed to combine in one reaction the analysis of several DNA orRNA targets, and the nucleic acid processing or analysis may involvemultiplexing of two or three enzymatic processes in one reaction.Furthermore, DNA and RNA enzymatic reactions frequently utilizesynthetic nucleic acid components, such as single stranded or doublestranded oligonucleotides that function as PCR or sequencing primers,ligation adaptors, or fluorescent probes, for example.

Adaptors and Their Use for DNA Processing

Supplementing DNA ends with additional short polynucleotide sequences,referred to as an adaptor or linker, is used in many areas of molecularbiology. The usefulness of adapted DNA molecules is illustrated by butnot limited to several examples, such as ligation-mediatedlocus-specific PCR, ligation-mediated whole genome amplification,adaptor-mediated DNA cloning, DNA affinity tagging, DNA labeling, etc.

Ligation-Mediated Amplification of Unknown Regions Flanking Known DNASequence

Libraries generated by DNA fragmentation and addition of a universaladaptor to one or both DNA ends were used to amplify (by PCR) andsequence DNA regions adjacent to a previously established DNA sequence(see U.S. Pat. No. 6,777,187 and references therein, for example, all ofwhich are incorporated by reference herein in their entirety). Theadaptor can be ligated to the 5′ end, the 3′ end, or both strands ofDNA. The adaptor can have a 3′ or 5′ overhang, depending on thestructure of the overhang generated by restriction enzyme digestion ofDNA. It can also a have blunt end, especially in the cases when DNA endsare “polished” after enzymatic, mechanical, or chemical DNAfragmentation. Ligation-mediated PCR amplification is achieved by usinga locus-specific primer (or several nested primers) and a universalprimer complementary to the adaptor sequence.

Ligation-Mediated Whole Genome Amplification

Libraries generated by DNA fragmentation and subsequent attachment of auniversal adaptor to both DNA ends were used to amplify whole genomicDNA (whole genome amplification, or WGA) (see U.S. patent applicationSer. Nos. 10/797,333 and 10/795,667 and references therein, for example,all of which are incorporated by reference herein in their entirety).The adaptor can be ligated to both strands of DNA or only to the 3′ endfollowed by extension. The adaptor can have a 3′ or 5′ overhang,depending on the structure of the DNA end generated by restrictionenzyme or other enzyme used to digest DNA. It can also have a blunt end,such as in the cases where DNA ends after enzymatic DNA cleavage areblunt or when the ends are repaired and “polished” after enzymatic,mechanical, or chemical DNA fragmentation. Whole genome PCRamplification is achieved by using one or two universal primerscomplementary to the adaptor sequence(s), in specific embodiments.

Adaptor-Mediated DNA Cloning

Adaptors (or linkers) are frequently used for DNA cloning (see Sambrooket al., 1989, for example). Ligation of double stranded adaptors to DNAfragments produced by sonication, nebulization, or hydro-shearingprocess followed by restriction digestion within the adaptors allowsproduction of DNA fragments with 3′ or 5′ protruding ends that can beefficiently introduced into a vector sequence and cloned.

Use of Stem-Loop (Hairpin) DNA Oligonucleotides for Nucleic AcidAnalysis

Stem-loop (also referred to as hairpin) oligonucleotides have been usedin several applications for analysis and detection of nucleic acids.These applications include molecular beacons, stem-loop PCR primers, andstem-loop DNA probes, immobilized on microarrays (Broude, 2002).

Molecular Beacons

A molecular beacon is a single-stranded oligonucleotide probe containinga sequence complementary to the target that is flanked byself-complementary termini, and carries a fluorophore and a quencher atthe 3′ and 5′ ends, respectively (Tyagi and Kramer, 1996). In theabsence of target the fluorophore and the quencher are in a closeproximity, which quenches fluorescence. Upon hybridization with thetarget, the beacon changes its conformation so that the fluorophore andthe quencher become separated, and fluorescence increases up to 100-200times. Molecular beacons have found many applications for real-timemonitoring of PCR (Tyagi et al., 1998) and isothermal amplification (deBaar et al., 2001), as microarray-immobilized probes (Liu et al., 2000),as antisense probes for RNA detection in vivo (Sokol et al., 1998), andas a probe to measure DNA polymerase activity (Summerer and Marx, 2002)and monitor conformational changes of DNA targets (Goddard et al.,2000).

Stem-Loop (Hairpin) PCR Primers

PCR primers containing hairpin structures on their 5′ ends with donorand acceptor moieties located in close proximity on the stem-loop stemhave been proposed for homogeneous (a closed tube) format foramplification, real-time quantification and mismatch detection by PCR(Broude, 2002). A stem-loop primer with a fluorophore at the 5′ end anda “scorpion” probe is simultaneously a molecular beacon and a PCR primer(Whitcombe et al., 1999). It has a tail attached to its 5′ end by alinker that prevents copying of the 5′ extension. The probe element isdesigned so that it hybridizes to its target only when the target sitehas been incorporated into the same molecule by extension of the tailedprimer. It was also shown that stem-loop probes can be used as primersfor PCR to reduce primer-dimer formation and mispriming, therebyincreasing its specificity (Kaboev et al., 2000).

Stem-Loop Microarray Probes

Stem-loop probes can also be used as capture devices if the loop isimmobilized on a surface and dangling ends are used forstacking-hybridization, thus providing both faster kinetics and higherhybrid stability (Riccelli et al., 2001) Immobilized molecular beaconprobes can be used for direct detection of non-amplified target DNA andRNA molecules (Hamad-Schifferli et al., 2002).

Multiplexed Reactions that Involve DNA and RNA Molecules

Several types of multiplexed reactions that involve DNA or RNA aredescribed to date. The multiplexed reactions can be divided into twomajor categories: reactions where two or more DNA/RNA sequences areamplified or detected simultaneously in one enzymatic process, andreactions where several enzymatic processes occur simultaneously withone DNA or RNA template.

Several Targets-One Enzyme

Multiplex PCR and RT-PCR are examples of the first category ofmultiplexed reactions (Mackay et al., 2000). In this case, severalgenomic or cDNA regions are amplified in one polymerase chain reactioncarried out by one thermostable DNA polymerase. Whole genome or wholetranscriptome amplification is another example of highly multiplexedDNA/RNA amplification reactions carried out by methophilic (Phi 29) orthermophylic (Taq) DNA polymerase (Sambrook et al., 1989).

One Target-Several Enzymes

“Long distance” PCR, nucleic acid sequence-based amplification (NASBA),its analog, transcription-mediated amplification (TMA), and DNAstrand-displacement amplification (SDA) are examples of the secondcategory of multiplexed DNA/RNA amplification reactions. In the “longdistance” PCR method there is a mixture of Taq DNA polymerase andanother thermo-stable DNA polymerase with 3′ proofreading activity(Barns™, 1994). TMA and NASBA methods utilize transcription-mediatedamplification that involves three enzymes: T7 RNA polymerase, reversetranscriptase, and RNase H (Deiman et al., 2001). In the SDA method, twoenzymes (a DNA polymerase and a restriction endonuclease) are combinedin one enzymatic step to amplify DNA (Hellyer and Naolean, 2004).

DNA nick-translation method is an example of a DNA labeling reactionthat involves two enzymes. The method is based on simultaneousincubation of DNA with DNase I and DNA polymerase I. DNase I generatesnicks in the DNA molecule, while DNA polymerase I incorporates labelednucleotides by initiating DNA synthesis from the nicked sites (Sambrooket al., 1989).

dU-glycosylase (which is also referred to as Uracyl-DNA Glycosylase orUDG) and endonuclease VIII can be combined to produce the enzymatic mix,or the USER enzyme for fragmentation of DNA containing dU bases (NewEngland Biolabs; Beverly, Mass.) may be employed. The fragmentationprocess occurs through enzymatic generation and cleavage of abasic sitesat positions of dU bases.

Several Targets-Several Enzymes

There is description of multiplexed amplification and detection ofseveral nucleic acid sequences using three-enzyme TMA and NASBA methods(van Deursen et al., 1999).

SUMMARY OF THE INVENTION

The present invention overcomes deficiencies in the art by providingnovel methods and compositions for amplification of a plurality ofdouble stranded DNA molecules by incorporating at least one sequenceonto the ends of the DNA molecules. However, the invention avoids thedisadvantages of other methods, such as the generation of primer dimersin polymerase chain reaction, for example.

In particular, the present invention allows for the amplification ofmolecules having at least one double stranded region by using adaptorsthat avoid the limitations of some adaptor molecules, such as thosehaving the propensity to form adaptor dimers. In certain aspects, thepresent invention provides an inert oligonucleotide for attachment to adouble stranded molecule such that it renders theoligonucleotide-ligated molecule capable of being modified, such asamplified, for example by polymerase chain reaction. Upon attachment ofthe inert adaptor to the molecule, the attached oligonucleotide becomesactive and suitable for providing at least in part one or more sequencesemployable for amplification, while the non-attached, free adaptorremains inactive. For example, during polymerase chain reaction thefree, non-attached inert adaptor can neither be primed nor used as a PCRprimer (until it is intentionally and specifically fragmented andconverted into a PCR primer); during amplification by transcription thepromoter sequence of the non-attached, free adaptor does not initiateRNA synthesis; during amplification by strand displacement, the nickingenzyme recognition sequence of the non-attached adaptor remains uncut.

In particular aspects, the inert adaptor comprises a stem-loopoligonucleotide, wherein the inertness of the stem-loop oligonucleotideis at least in part a result of the dormant nature of its uniquestructure, biochemical properties (one enzymatically active end), and/orphysico-chemical characteristics (extremely high thermostability, forexample). The oligonucleotide may comprise RNA, DNA, or both. Theoligonucleotide may have one or more hairpins, and it may be furtherdescribed as comprising any structure with multiple secondary structureand only one end. In specific embodiments, the oligonucleotide canchange its functionality upon attachment to a double stranded nucleicacid molecule (DNA, RNA, or both). In other words, because of aconformational change of the oligonucleotide upon (such as following,for example) attachment to a double stranded nucleic acid molecule, oneor more functional properties of the oligonucleotide become altered,such as one or more functional properties hidden in the oligonucleotideprior to attachment manifesting upon attachment.

In addition to the advantages provided by the adaptors of the invention,the invention further provides novel conditions for modification of DNAmolecules with the adaptors, and subsequent amplification. In most casesin the art, the process of DNA analysis involves multiple enzymaticreactions that are performed in a sequential manner, frequently withintermediate purification steps between at least some of the reactions.For example, preparation of DNA libraries for subsequent amplificationand analysis involves ligation of adaptor sequences to DNA ends tointroduce a priming site for the PCR-mediated whole genome amplificationand ligation-mediated PCR, a promoter element for thetranscription-mediated DNA amplification, and/or cohesive ends tofacilitate DNA integration into a vector molecule for cloning.Preparation of library from genomic or viral DNA by current adaptorattachment procedures typically occur in 6 steps (FIG. 1A):

Step 1—fragmentation of high molecular weight (HMW) DNA to a sizeamenable to amplification or cloning;

Step 2—DNA purification to remove nucleases and other reagents;

Step 3—“polishing” of DNA ends by a 3′ proofreading DNA polymerase(s) togenerate blunt-ended DNA fragments with the 3′ hydroxyl and 5′ phosphategroups at the termini;

Step 4—DNA purification to remove the polymerase and replace the buffer,or, alternatively, heat inactivation of the DNA polymerase;

Step 5—adaptor ligation; and

Step 6—3′ end extension (in the case when an adaptor is ligated only toone strand of DNA, specifically to the 5′ phosphate).

However, such a multi-step process takes a considerable amount of time(1-2 days) and can be a major obstacle for high throughput anddiagnostic-type applications, for example. The process becomes morecomplicated when library preparation involves additional enzymaticsteps, such as DNA or library digestion with methylation-sensitive ormethylation-specific endonucleases (for example, in the preparation ofMethylome libraries, such as are described in U.S. patent applicationSer. No. 11/071,864, filed Mar. 3, 2005, which is incorporated byreference herein in its entirety), or restriction nuclease cleavagewithin the adaptor sequence to produce sticky ends for DNA cloning.

The present invention satisfies a long-felt need in the art forobviating the requirement for multiple manipulations for nucleic acidprocessing. The current invention introduces and demonstrates newmethods and compositions that allow reduction of several importantmulti-step enzymatic DNA processes to a single-step-reaction that isperformed in one reaction volume (FIGS. 1B and 1C). Such simplificationhas been achieved by developing a highly multiplexed enzymatic method(Enz-O-Mix) that combines a substantial number (from 2 to 10) ofenzymatic processes into one complex reaction mixture that occurs underuniversal buffer conditions.

Specifically, the current invention introduces novel processes of DNAlibrary preparation and/or DNA amplification (FIG. 1C) that reduce thewhole multi-step process to a single-step multiplexed enzymatic reactioninvolving as a minimum two Enzymes, a stem-loop (hairpin)Oligonucleotide with a special base/bonds composition, and a universalbuffer (Mix), which is herein referred to as Enz-O-Mix, that supportsefficient functioning of all enzymatic activities present in themixture. The invention demonstrates that the Enz-O-Mix approach can beused to prepare a DNA library for whole genome amplification (WGALibrary), a Methylome library for amplification and analysis ofmethylated DNA regions, and/or even directly amplify DNA in a singlemulti-enzyme reaction, for example.

The Enz-O-Mix library/amplification has no limitations on the size andnature of DNA in the reaction. The method can be applied to highmolecular weight DNA, such as is isolated from tissues or cell culture,for example, as well as highly degraded DNA, such as cell-free DNA fromblood and urine and/or DNA extracted from formalin-fixed,paraffin-embedded tissues, for example.

The applications of the Enz-O-Mix method are numerous. The Enz-O-Mixmethod is easy to automate and use in clinical diagnostic andpoint-of-care applications, for example. In specific embodiments,different enzyme combinations are utilized for many novel kits andassays and utilized in such areas as biotechnology, the pharmaceuticalindustry, molecular diagnostics, forensics, pathology, bio-defense,and/or bio-computing, for example. The Enz-O-Mix approach is a simpleand cost-effective alternative to the “lab-on-a chip” microfluidicapproach that is currently attempting to solve the same problem(multi-step DNA processing) by reduction of reaction volumes andintegration of multiple reactions into a small format (FIG. 2). Themethod can be used for in vitro as well as for in vivo nucleic acidamplification and/or detection.

In specific aspects, the present invention introduces a concept ofmultiplexing two or more enzymatic processes in one reaction, teacheshow to optimize a highly multiplexed enzymatic process, and demonstratesin multiple specific examples the efficacy of this approach. Inparticular embodiments, the present invention is directed tocompositions and methods for simultaneous processing of DNA moleculeswith a combination of enzymes in a one-step-one-tube reaction andproducing either a collection of molecules suitable for amplification,or amplified DNA molecules.

In particular, the present invention greatly reduces the number of stepsfor library preparation by consolidating a series of steps into onestep. For known whole genome amplification or whole methylome methods,these protocols greatly reduce the number of steps for library synthesesthat utilize adaptors. That is, in the case of WGA library preparationfrom high molecular weight DNA, for example, the number of steps isreduced from about 6 to 1 (FIGS. 1A and 1B). In the case of WMA librarypreparation from high molecular weight DNA, for example, the number ofsteps is reduced from about 7 to 1. In the case of WGA librarypreparation from fragmented DNA (plasma, serum, or urine, for example),the number of steps is reduced from about 4 to 1 (FIG. 1B). In the caseof Methylome library preparation from fragmented DNA, for example, thenumber of steps is reduced from about 5 to 1 (FIG. 1B).

In particular embodiments, the methods of the invention can be easilyapplied to any type of fragmented double stranded DNA including but notlimited to, for example, free DNA isolated from plasma, serum, and/orurine; apoptotic DNA from cells and/or tissues; DNA fragmentedenzymatically in vitro (for example, by DNase I and/or restrictionendonuclease); and/or DNA fragmented by mechanical forces (hydro-shear,sonication, nebulization, etc.).

In other embodiments, the invention can be easily applied to any highmolecular weight double stranded DNA including, for example, DNAisolated from tissues, cell culture, bodily fluids, animal tissue,plant, bacteria, fungi, viruses, etc.

In one embodiment of the invention, there is incubation of DNA with anenzymatic mixture (Enz-O-Mix) comprising from about 2 to about 18different enzymes in one buffer and a stem-loop (hairpin)oligonucleotide with a specific base/bonds composition.

In particular embodiments, there is a one-step enzymatic processincluding one or more of the following: a) “polishing” of DNA andstem-loop oligonucleotide ends by Klenow fragment of DNA polymerase I(or other proofreading DNA polymerase); b) ligation of the 3′ end ofstem-loop oligonucleotide to the 5′ end of DNA, such as by a ligase, forexample, by T4 DNA ligase; c) “fill-in” DNA synthesis that is initiatedat the 3′ end of DNA, propagates towards the end of the stem-loopoligonucleotide, and stops at a replication block or at the end of theoligonucleotide; and d) cleavage in the middle of a generated invertedrepeat by a restriction nuclease; and e) restriction cleavage with amixture of methylation-sensitive restriction enzymes ormethylation-specific nuclease(s) (such as in embodiments concerningwhole methylome amplification library), for example.

In particular aspects of the invention, an inverted repeat (palindrome)at the end of DNA molecules is not generated (or is generated andremoved), because it may inhibit the amplification step (PCR), primingof the cDNA strand synthesis (amplification by transcription), orpriming of the second DNA strand synthesis (amplification by stranddisplacement DNA synthesis). Thus, in specific embodiments at least partof the oligonucleotide sequence is eliminated, such as, by terminationof replication in the middle of a stem-loop oligonucleotide or bycutting the generated inverted repeat internally, for example.

In one specific embodiment of this invention, a chemical modification,such as hexaethylene glycol (HEG) linker, a bulky base analog, or one orseveral abasic sites within the loop or adjacent to it, can beintroduced during oligonucleotide synthesis. Such modifications willterminate the replication within the stem-loop oligonucleotide.

In another specific embodiment, the replication block is generatedduring the reaction by incorporating one or more dU bases into thestem-loop oligonucleotide design and including dU-glycosylase (which isalso referred to as Uracyl-DNA Glycosylase or UDG) in the reaction mix.

In some embodiments, a specific site for a cleavage enzyme is generatedduring “fill-in” DNA synthesis that is initiated at the 3′ end of theDNA, propagates towards the end of the stem-loop oligonucleotide, andstops at the 5′ end of the oligonucleotide. Such a site residesoriginally either completely or partially within the single-strandedloop of an oligonucleotide and becomes functional (cleavable) only afterconversion of this loop into double-stranded form.

In one specific embodiment a cleavage enzyme is represented by arestriction endonuclease, in another by a homing endonuclease.

In some embodiments, the methods further comprise simultaneous digestionwith an endonuclease or combination of endonucleases, such asrestriction endonucleases, DNase I, benzonase, methylation-specificnuclease McrBC, apoptotic endonucleases, etc., to generate in one stepWhole Genome or Whole Methylome library even from high molecular weight(HMW) DNA.

In particular embodiments, a library generated by a one-step process isfurther amplified by PCR using universal primer complementary to thesequence introduced by attachment of a stem-loop oligonucleotide.

In particular embodiments, library generation and PCR amplification arecombined into one integrated, closed-tube process in a thermocycler.

In one particular embodiment, an integrated, closed-tube librarypreparation/amplification process is supported by a universal PCR primerincluded in the original Enz-O-Mix.

In another particular embodiment, an integrated, closed-tube librarypreparation/amplification process is supported by a stem-loopoligonucleotide converted into a functional universal primer as a resultof enzymatic and chemical reactions that occur before PCR amplification.

In another embodiment, degradable stem-loop oligonucleotides are used aslocus-specific primers for a hot start PCR process.

In another particular embodiment, a library generated by one-stepprocess is further amplified by transcription utilizing a promotersequence (T7, T3, SP6, etc.) introduced by attachment of a stem-loopoligonucleotide.

In particular embodiments, a stem-loop oligonucleotide design isemployed for the one-step library synthesis reaction, although inalternative embodiments other adaptors may be utilized, such as astandard linear two-oligonucleotide adaptor, for example.

In particular embodiments, a stem-loop oligonucleotide provides asynthesized library with a new function that was present neither in theDNA molecule nor in the stem-loop oligonucleotide prior to itsattachment. Examples include a functionally inactive T7 promotersequence, a non-cleavable restriction site, and/or inactive nicking siteresiding at least partially within the loop or stem region of thestem-loop oligonucleotide and activated only by the attachment processand conversion of the single stranded loop into a double strandedmolecule.

In particular embodiments, a stem-loop oligonucleotide provides asynthesized library with a 5′ overhang that was not present either inthe DNA molecule or in the stem-loop oligonucleotide prior to itsattachment, and that can be used for covalent or non-covalentimmobilization of a synthesized library on a solid support viahybridization or hybridization and ligation to a covalently attachedoligonucleotide.

In a specific embodiment, the library synthesis and its immobilizationwithin the tube (well) occurs simultaneously in a one Enz-O-Mix process.In this case, combinations of enzymes and stem-loop oligonucleotides aremixed together in a special tube with a covalently attached captureoligonucleotide to produce Enz-O-Mix-Immobilization kits for highthroughput DNA processing and diagnostic applications in solid phaseformat.

In particular embodiments, combinations of enzymes and stem-loopoligonucleotides are mixed together to produce Enz-O-Mix kits for highthroughput DNA processing and diagnostic applications. In particularembodiments, there is a Universal Enz-O-Mix Buffer that providessimultaneous activity of all enzymes used in the reaction.

In particular embodiments, combinations of enzymes and stem-loopoligonucleotides are mixed together to produce integrated, closed-tubeEnz-O-Mix kits for high throughput DNA fragmentation, amplification andlabeling. In particular embodiments, there is a Universal Enz-O-MixBuffer that supports simultaneous activities of all enzymes used in thereaction.

In a specific embodiment, there is a process for optimization of theUniversal Buffer and other components of the multiplexed enzymaticreaction.

In particular aspects, the present invention is directed to a system andmethod for preparing a collection of molecules, particularly moleculessuitable for amplification, such as amplification utilizing knownsequences on the molecules. In specific embodiments, the oligonucleotidecomprises a known sequence.

In an additional embodiment, there is a kit housed in a suitablecontainer that comprises one or more compositions of the inventionand/or comprises one or more compositions suitable for at least onemethod of the invention.

In one embodiment of the invention, there is a method of preparing anucleic acid molecule, comprising: providing a double stranded nucleicacid molecule; and attaching one strand of a stem-loop oligonucleotidecomprising an inverted repeat and a loop to the double stranded nucleicacid molecule to produce an oligonucleotide-attached nucleic acidmolecule. The double stranded nucleic acid molecule may be a doublestranded DNA molecule, in some embodiments. In specific embodiments, theattaching is further defined as attaching the oligonucleotide to thedouble stranded nucleic acid molecule under conditions to produce anon-covalent junction, such as a nick, a gap, or a 5′ flap structure, inthe oligonucleotide-attached nucleic acid molecule. In particularaspects of the invention, the attaching is further defined as ligating.The method may further comprise displacing one strand of theoligonucleotide from the oligonucleotide-attached nucleic acid moleculeby strand displacement or by nick translation polymerization. In aspecific embodiment, at least part of the oligonucleotide-attachednucleic acid molecule is amplified, such as by polymerase chainreaction, RNA transcription, or strand displacement, for example.Methods of the invention may further comprise amplifying anoligonucleotide-attached nucleic acid molecule, wherein at least part ofthe inverted repeat is excluded from the amplifiedoligonucleotide-attached nucleic acid molecule.

Ligating embodiments may be further defined as comprising: generatingligatable ends on the double stranded nucleic acid molecule; generatinga ligatable end on the stem-loop oligonucleotide; and ligating onestrand of the ligatable end of the stem-loop oligonucleotide to onestrand of an end of the nucleic acid molecule, thereby generating anon-covalent junction, such as a nick, a gap, or a 5′ flap structure, inthe oligonucleotide-attached nucleic acid molecule. In further aspects,the methods comprise generating blunt ends on the nucleic acid molecule;generating a blunt end on the stem-loop oligonucleotide; and ligatingone strand of the blunt end of the stem-loop oligonucleotide to onestrand of a blunt end of the nucleic acid molecule, thereby generating anick in the oligonucleotide-ligated nucleic acid molecule.

In specific aspect of the invention, the stem-loop oligonucleotidecomprises a known sequence, for example a regulatory sequence, includinga RNA polymerase promoter sequence. A regulatory sequence may reside atleast in part within the stem of the stem-loop oligonucleotide, withinthe loop of the stem-loop oligonucleotide, or both.

Methods of the invention may further comprise digesting the DNA moleculewith one or more endonucleases to produce DNA fragments; producing bluntends on the DNA fragments; producing a blunt end on the stem-loopoligonucleotide; and ligating one strand of the blunt end of a stem-loopoligonucleotide to one strand of a blunt end of a DNA fragment, therebygenerating a nick in an oligonucleotide-ligated DNA fragment. In aspecific embodiment, the endonuclease is a restriction endonuclease,DNAse I, or an apoptotic endonuclease, or a mixture thereof. Inparticular embodiments, the restriction endonuclease ismethylation-specific or methylation-sensitive.

In additional embodiments, the oligonucleotide-attached nucleic acidmolecule comprises a nick having a 3′ hydroxy group, wherein there ispolymerization from the 3′ hydroxy group of at least part of theoligonucleotide-attached nucleic acid molecule.

Strand displacement or nick translation polymerization may be furtherdefined as polymerization that ceases at a non-replicable base or regionin the loop or in a region of the stem adjacent to the loop.

In a specific aspect of the invention, the method further comprises thestep of digesting the double stranded DNA molecule with an endonucleaseto generate DNA fragments, wherein the oligonucleotide becomes ligatedto one strand of the DNA fragment and wherein polymerization of anoligonucleotide-ligated DNA fragment excludes at least part of theinverted repeat by subjecting the oligonucleotide-ligated DNA fragmentto strand displacement or nick translation polymerization that halts ata base or sequence in the loop or in a region of the stem adjacent tothe loop.

In some embodiments, the stem-loop oligonucleotide is further defined ascomprising a non-replicable base or sequence. In particular, in somecases at least part of the non-replicable base or sequence is present inthe loop of the oligonucleotide or in a sequence of the stem adjacent tothe loop. The non-replicable base or sequence may comprise an abasicsite or sequence, hexaethylene glycol, and/or a bulky chemical moietyattached to the sugar-phosphate backbone or the base. In specificembodiments, the abasic site or sequence is introduced by one or moreenzymes in the single solution. In a further specific embodiment, theloop of the stem-loop oligonucleotide comprises at least onedeoxy-uridine.

In one particular embodiment, strand displacement or nick translationpolymerization of the oligonucleotide-attached nucleic acid moleculegenerates an endonuclease site. In particular aspects, the methodsfurther comprise the step of digesting the double stranded DNA moleculewith an endonuclease to generate DNA fragments, wherein theoligonucleotide becomes blunt end ligated to one strand of the DNAfragment to produce an oligonucleotide-ligated DNA fragment, and whereinstrand displacement or nick translation polymerization of theoligonucleotide-ligated DNA fragment generates an endonuclease site.

In particular aspects, the endonuclease site is a site-specificrestriction endonuclease site and at least part of the inverted repeatis removed by cleavage with said restriction endonuclease. Theendonuclease site may reside within the stem and/or loop regions.Examples of endonucleases include Eco NI or PacI. The endonuclease sitemay be a homing endonuclease site, such as I-Ceu I, I-Sce I, Pl-Psp I,or Pl-Sce I. In specific embodiments, RNA transcription is initiatedfrom the regulatory sequence, thereby producing at least one transcribedpolynucleotide.

In specific embodiments, a regulatory sequence resides at least in partwithin the stem of the stem-loop oligonucleotide, or at least in partwithin the loop of the stem-loop oligonucleotide. In a specific aspect,the regulatory sequence comprises a bacteriophage regulatory sequence,such as a T7 regulatory sequence, a T3 regulatory sequence, or a Sp6regulatory sequence, for example. In specific aspects, a transcribedpolynucleotide is replicated by a reverse transcriptase, which may beinitiated by hybridization of the oligonucleotide complementary to the3′ end of the transcribed polynucleotide.

The stem-loop oligonucleotide may further comprise a recognitionsequence for a nicking endonuclease and the oligonucleotide-attachednucleic acid molecule is amplified by strand displacement synthesis andsecond strand synthesis, in particular aspects of the invention. In aspecific aspect, the recognition sequence for a nicking endonucleaseresides at least in part within the stem of the stem-loopoligonucleotide. The recognition sequence for a nicking endonuclease mayreside at least in part within the loop of the stem-loopoligonucleotide. In specific embodiments, the nicking endonuclease isN.Alw I, N.Bbv CIA, N.Bbv CIB, Nb.Bpu10I, Nb.BsmI, N.BstNBI, or N.Bst9I.

In specific aspects, a 5′ end of the stem-loop oligonucleotide lacks aphosphate.

In particular embodiments, the oligonucleotide-attached nucleic acidmolecule is further modified, such as by cloning, including byincorporation of the modified molecule into a vector, said incorporationoccurring at ends in the modified molecule generated by endonucleasecleavage within the inverted repeat.

In additional embodiments of the invention, methods of the inventionoccur in a single suitable solution, wherein the process occurs in theabsence of exogenous manipulation. The method may occur at onetemperature, such as from about 10° C. to about 75° C. In particularembodiments, the solution comprises one or more of the following:ligase; DNA polymerase; one or more endonucleases; RNA polymerase;reverse transcriptase; RNase H; deoxy-uridine glycosylase (which is alsoreferred to as Uracyl-DNA Glycosylase or UDG); nickase; thermophilic DNApolymerase; ATP; rNTPs; and dNTPs.

In one aspect of the invention, there is a kit, housed in a suitablecontainer, comprising: a stem-loop oligonucleotide; and a solutionsuitable for ligation of the oligonucleotide onto a double strandedmolecule; and, optionally, one or more of the following: ligase; DNApolymerase one or more restriction enzymes; RNA polymerase; reversetranscriptase; RNase H; nickase; thermophilic DNA polymerase; ATP;rNTPs; and dNTPs. In specific embodiments, the oligonucleotide of thekit comprises a RNA polymerase promoter sequence, such as a T7 RNApolymerase promoter sequence, for example. The one or more endonucleasesmay be methylation-specific, methylation-sensitive, or may be a homingendonuclease.

In specific embodiments, methods of the invention occur in a singlesuitable solution, wherein the process of preparing and amplifying of aoligonucleotide-attached nucleic acid molecule occurs in the absence ofexogenous manipulation. The method may be further defined as occurringat one temperature, such as between about 10° C. to about 85° C. orbetween about 10° C. to about 85° C. In specific embodiments, the methodis further defined as occurring at least two different temperatures,such as wherein at least one of the temperatures is from about 10° C. toabout 100° C.

In specific embodiments, the oligonucleotide-attached nucleic acidmolecule is immobilized to a solid support, such as non-covalently orcovalently.

Some methods of the invention further comprise the step of digesting theDNA molecule with an endonuclease to generate DNA fragments, wherein theoligonucleotide becomes attached to one strand of the DNA fragment,wherein strand displacement polymerization of theoligonucleotide-ligated DNA fragment and its arrest at a base orsequence in the loop or in a region of the stem adjacent to the loopgenerates a 5′ overhang, and wherein the single stranded 5′ overhanghybridizes to a complementary oligonucleotide covalently immobilized toa solid support.

Additional embodiments of the invention include a library of DNAmolecules prepared by the methods of the invention.

In some embodiments of the invention, there is a method of preparing aDNA molecule, comprising: providing an oligonucleotide; and mixing theoligonucleotide with a double stranded DNA molecule, such that uponattachment of the oligonucleotide to the double stranded DNA molecule,the oligonucleotide attached to the DNA molecule is capable ofdemonstrating a function that it was incapable of before the attachmentto the double stranded DNA molecule, thereby producing anoligonucleotide-attached DNA molecule suitable for modification. Inspecific aspects, the method occurs in a single suitable solution,wherein the process occurs in the absence of exogenous manipulation.Exemplary modifications include site-specific nicking, site-specificdouble-strand cleavage, transcription, recombination, amplification,polymerization, nick translation, strand displacement, immobilization ora combination thereof.

In an additional embodiment of the invention, there is a compositioncomprising a stem-loop oligonucleotide, said oligonucleotide comprisingan inverted repeat, a loop and at least one thermo-sensitive site orsite capable of becoming a thermo-sensitive site, wherein theoligonucleotide is capable of producing a suitable primer upon exposureto heat. In additional embodiments, the composition further comprises arepair enzyme, such as one capable of converting damaged bases into anabasic site. Examples of DNA repair enzymes include a DNA glycosylase,such as dU-glycosylase, for example. In a specific aspect, thethermo-sensitive site is an abasic site, such as an apyrimidine site oran apurine site, for example. In an additional specific embodiment, thesite capable of becoming a thermo-sensitive site is further defined as abase that can be enzymatically converted to a thermo-sensitive site. Thebase that can be enzymatically converted to a thermo-sensitive site maybe deoxy-uridine and the enzyme may be dU-glycosylase (which is alsoreferred to as Uracyl-DNA Glycosylase or UDG). In a specific aspect, thethermo-sensitive site is introduced into the oligonucleotide duringsynthesis, such as chemical synthesis. The composition may be capable ofcomprising a breakage of a strand of the oligonucleotide at one or moredeoxy-uridine bases upon exposure to heat. The thermosensitive site orsite capable of becoming a thermo-sensitive site may be located in thestem, in the loop, or both. The oligonucleotide may be blunt ended orhave a 5′ overhang.

In an additional embodiment, there is a method of producing a primer,comprising providing a composition of the invention and subjecting thecomposition to heat such that the primer is produced. In a specificaspect, the composition further comprises a DNA repair enzyme and thesubjecting to heat is further defined as subjecting to two or moreexposures of heat. One exposure to heat may comprise about 37° C. andanother exposure to heat may comprise about 95° C. One exposure to heatmay comprise about 10° C. to about 100° C. whereas another exposure toheat may comprise about 80° C. to about 100° C. The method may furthercomprise amplification of a DNA molecule utilizing said composition.

The foregoing has outlined rather broadly the features and technicaladvantages of the present invention in order that the detaileddescription of the invention that follows may be better understood.Additional features and advantages of the invention will be describedhereinafter which form the subject of the claims of the invention. Itshould be appreciated by those skilled in the art that the conceptionand specific embodiment disclosed may be readily utilized as a basis formodifying or designing other structures for carrying out the samepurposes of the present invention. It should also be realized by thoseskilled in the art that such equivalent constructions do not depart fromthe spirit and scope of the invention as set forth in the appendedclaims. The novel features that are believed to be characteristic of theinvention, both as to its organization and method of operation, togetherwith further objects and advantages will be better understood from thefollowing description when considered in connection with theaccompanying figures. It is to be expressly understood, however, thateach of the figures is provided for the purpose of illustration anddescription only and is not intended as a definition of the limits ofthe present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the present invention, reference isnow made to the following descriptions taken in conjunction with theaccompanying drawings.

FIG. 1A shows a standard multi-step DNA adaptor attachment process.

FIG. 1B compares preparation processes for WGA and WMA libraries fromdegraded serum and urine DNA described in the present invention and inpreviously filed patent applications (U.S. patent application Ser. No.11/071,864, filed Mar. 3, 2005).

FIG. 1C is a general representation of the invention that can bedescribed as a reduction of several important multi-step enzymatic DNAprocesses to a single-step multiplexed enzymatic reaction that isperformed in one reaction volume (Enz-O-Mix Method).

FIG. 2 shows that the Enz-O-Mix approach can be viewed as a simple andcost-effective alternative to the “lab-on-a chip” microfluidic approachthat is currently attempting to solve the same problem by reduction ofreaction volumes and integration of multiple reactions into a smallformat.

FIG. 3 is a schematic description and composition of specific componentsof the Enz-O-Mix method and reagents involved in the attachment ofstem-loop oligonucleotides to DNA ends. Four enzymatic reactions aretaking place nearly simultaneously: “polishing” of the DNA ends and thehairpin double-stranded stem-region; ligation of the stem-loopoligonucleotide 3′ end to the 5′ phosphate of the DNA, leaving a nickbetween the 3′ end of DNA and the 5′ end of the hairpin double-strandedstem-region; polymerase extension of the 3′ DNA end that propagatestoward the end of stem-loop oligonucleotide; and by strand-displacementreaction within the oligonucleotide stem region. The process results inthe library of DNA fragments with inverted repeat sequences at theirends.

FIG. 4 shows three original secondary structures of the stem-loopoligonucleotide with the 3′ or 5′ protruding, or blunt end, and itsfinal (blunt end) structure within the Enz-O-Mix.

FIGS. 5A-5B are schematic descriptions of the amplification of a libraryof DNA fragments with inverted repeat sequences at their ends by RNAtranscription. Promoter sequence can be added to a DNA end either as apart of the oligonucleotide stem-region (A), or as a part of theoligonucleotide loop region (B). The products of the amplification areRNA molecules in this embodiment.

FIG. 6A illustrates the components and enzymatic reactions involved inthe one-step DNA amplification by transcription using Enz-O-Mix reagent(Enz-O-Mix 5 in FIG. 19B, for example). The process linearly amplifiesDNA and produces single stranded RNA molecules.

FIG. 6B illustrates additional components and enzymatic reactionsinvolved in the one-step DNA amplification by transcription accompaniedby the synthesis of complementary cDNA strand. The transcription(replication) block (see Sections E-H and FIGS. 8-10) is introduced intothe stem-loop oligonucleotide upstream of the T7 promoter region toprevent formation of an inverted repeat at the 3′ end of RNA moleculesand thus increase the efficiency of the oligo T7 priming (thetranscription block is shown as a black square). The process canlinearly amplify DNA and produce double stranded DNA/RNA hybrids.

FIG. 7 is an illustration of the inhibitory effect on PCR of invertedrepeats attached to both ends of DNA fragments. Heating and replicationgenerates DNA molecules refractory to melting, priming, and PCRamplification either using the universal primer A or any internalsite-specific primer pair.

FIG. 8 is a schematic description of the one-step Enz-O-Mix attachmentprocess for a stem-loop oligonucleotide with non-replicable linker. Thefollowing enzymatic reactions are taking place nearly simultaneously:“polishing” of the DNA ends and the stem-loop oligonucleotidedouble-stranded stem-region; ligation of the oligonucleotide 3′ end tothe 5′ phosphate of the DNA, leaving a nick between the 3′ end of DNAand the 5′ end of the hairpin double-stranded stem-region; polymeraseextension of the 3′ DNA end that propagates toward the end ofoligonucleotide and stops at the replication block (non-replicablelinker) within the loop or outside the loop but no more than about sixbases away from the loop. The process results in the library of DNAfragments with universal sequence A at the ends and an inverted repeatattached only to the 5′ end of DNA.

FIG. 9 is an illustration of the absence of an inhibitory effect on PCRof inverted repeats attached to only 5′ ends of DNA fragments. Heatingand replication generates DNA molecules with the universal sequence Aand no inverted repeats at the ends (shown by dash lines) that can besuccessfully amplified by PCR.

FIG. 10A shows the structure of a stem-loop oligonucleotide withnon-replicable linker introduced chemically during oligonucleotidesynthesis and detailed events occurring at a DNA end during themulti-enzyme attachment process. The process occurs as described in FIG.8.

FIG. 10B shows the structure of a stem-loop oligonucleotide withnon-replicable linker introduced enzymatically during the attachmentreaction and detailed events occurring at a DNA end during themulti-enzyme attachment process. Specifically, dU-glycosylase generatesabasic sites within the loop and upper strand of the stem region of thestem-loop oligonucleotide. The abasic sites within a loop generate anon-replicable region, while the sites in a stem destabilize the duplexand facilitate strand displacement reaction. The process results in alibrary of DNA fragments with universal sequence at the ends and aninverted repeat attached only to the 5′ end of DNA. Heating at 95° C.during PCR generates breaks at abasic sites and completely eliminatesthe 5′ portion of a stem-loop oligonucleotide.

FIG. 11A shows a standard adaptor formed by two differentoligonucleotides, wherein one of them has a 5′ phosphate group, andpossible products formed when such adaptor is used in the multi-enzymeone-step ligation process.

FIG. 11B shows a standard adaptor formed by two differentoligonucleotides without phosphate group and two products formed whensuch an adaptor is used in the multi-enzyme one-step ligation process.Only 50% of formed molecules are desirable products with correctorientation of the adaptor, whereas the other 50% of molecules haveinverse adaptor orientation.

FIG. 11C shows a standard adaptor formed by two differentoligonucleotides without a phosphate group, a protective group at the 3′end of one oligonucleotide, and products formed when such an adaptor isused in the multi-enzyme one-step ligation process. As in the casedescribed on FIG. 11B, only 50% of formed molecules are desirableproducts with correct orientation of the adaptor, whereas the other 50%of molecules have inversed adaptor orientation.

FIG. 12 is a schematic description of the one-step Enz-O-Mix attachmentprocess for a stem-loop oligonucleotide with an enzymatically cleavablesite generated during the multi-enzyme reaction. Specifically, theendonuclease recognizes and cuts the oligonucleotide specificoligo-sequence R when it adopts a double-stranded conformation as aresult of the attachment process. The reaction results in a library ofDNA fragments with universal sequence A at the ends and no invertedrepeat attached to the ends of DNA fragments.

FIG. 13A shows the stem-loop oligonucleotide with the recognitionsequence for the restriction endonuclease Eco NI located at the loopregion. The enzyme can not cut the CCTNNNNNAGG (SEQ ID NO: 20) regionwithin the stem-loop oligonucleotide (not a recognizable structure) butit can cut it efficiently when the oligonucleotide is attached to a DNAend and adopts a canonical (double-stranded) Watson-Crick conformation.

FIG. 13B shows the stem-loop oligonucleotide with the recognitionsequence for the restriction endonuclease Pad located within the loopregion. The enzyme can not cut the single stranded TTAATTAA regionwithin the stem-loop oligonucleotide, but it cuts it efficiently whenthe oligonucleotide is attached to a DNA end and adopts a canonical(double-strand) Watson-Crick conformation.

FIG. 13C shows the stem-loop oligonucleotide with the recognitionsequence for the exemplary homing endonuclease I-Ceu I located withinthe loop region. The enzyme can not cut the 26-base single-strandedregion within the hairpin loop, but it cuts efficiently when theoligonucleotide is attached to a DNA end and adopts a canonical(double-stranded) Watson-Crick conformation.

FIG. 14 is a schematic description of the one-step Enz-O-Mix attachmentprocess for a stem-loop oligonucleotide with a very short stem. Theprocess results in the library of DNA fragments with universal sequenceA at the ends and very short inverted repeat attached to the ends of DNAfragments. Amplification by PCR is possible, because the meltingtemperature for terminal hairpins is low enough to prevent folding,self-priming and formation of long hairpin molecules, as shown on FIG.7.

FIG. 15 illustrates the components and enzymatic reactions involved inexemplary one-step or two-step (after library synthesis) DNAamplification by strand displacement DNA synthesis using Enz-O-Mixreagents (Enz-O-Mix 3 and Enz-O-Mix 6 in FIGS. 19A and 19B, forexample). The amplification is initiated and further maintained by thegeneration of a nick within the attached oligonucleotide sequence. Theprocess linearly amplifies DNA and produces double stranded DNAmolecules.

FIG. 16A shows the stem-loop oligonucleotide with the recognitionsequence for the nicking endonuclease N. Bbv CIA located at the loopregion. The enzyme can not cut the single stranded GCTGAGG region withinthe stem-loop oligonucleotide (not a recognizable structure) but it cannick it efficiently when the oligonucleotide is attached to a DNA endand adopts a canonical (double-stranded) Watson-Crick conformation.

FIG. 16B shows a stem-loop oligonucleotide with the recognition sequencefor the nicking endonuclease N. Bbv CIA located at the end of a stemregion. The enzyme can not nick efficiently the GCTGAGG region withinthe stem-loop oligonucleotide (it is too close to the end) but it cannick it efficiently when the oligonucleotide is attached to a DNA end;the second site generated by attachment is not a good substrate fornicking (it is too close to the end).

FIG. 16C shows two exemplary multifunctional stem-loop oligonucleotideswith several regulatory elements within a single (1) or multiple (2)loop regions: T7 promoter sequence (that includes all regions necessaryfor transcription), recognition sequence N for a nicking endonuclease,recognition sequence for a restriction endonuclease, and areplication/transcription block region (a chemical modification or astring of 2-4 dU bases). A multifunctional stem loop oligonucleotide cansupport different types of DNA amplification, including PCR, isothermalamplification by transcription, isothermal amplification by stranddisplacement synthesis, or even a combination thereof and it can alsofacilitate other modifications, including DNA immobilization, DNArecombination, DNA cloning, and a combination thereof, for example.

FIG. 17 shows generalization of the Enz-O-Mix method and demonstratesspecific but exemplary applications.

FIG. 18 shows composition of five exemplary different Stem-Loop OligoAttachment Master Mixes of the invention that are used for the one-stepnucleic acid library synthesis.

FIG. 19A schematically shows compositions of several Enz-O-Mix reagents,specifically, Enz-O-Mix 1, designed to convert fragmented DNA into aWhole Genome Library that can be amplified by PCR in an open, orclose-tube format. Enz-O-Mix 2 and Enz-O-Mix 3 are designed to convertfragmented DNA into a Whole Genome Library that can be amplified afteror during the Whole Genome Library synthesis by transcription and stranddisplacement, respectively.

FIG. 19B shows schematically compositions of several Enz-O-Mix reagents,specifically, Enz-O-Mix 4, designed to convert HMW DNA into a WholeGenome Library that can be amplified by PCR in an open, or close-tubeformat. Enz-O-Mix 5 and Enz-O-Mix 6 are designed to convert fragmentedDNA into a Whole Genome Library that can be amplified after or duringthe Whole Genome Library synthesis by transcription and stranddisplacement, respectively.

FIG. 19C shows schematically compositions of several Enz-O-Mix reagents,specifically, Enz-O-Mix 7, designed to convert fragmented DNA into aWhole Methylome Library that can be amplified by PCR. Enz-O-Mix 8 andEnz-O-Mix 9 are designed to convert fragmented DNA into a WholeMethylome Library that can be amplified after or during the WholeMethylome Library synthesis by transcription and strand displacement,respectively.

FIG. 19D shows schematically compositions of several Enz-O-Mix reagents,specifically, Enz-O-Mix 10, designed to convert HMW DNA into a WholeMethylome Library that can be amplified by PCR. Enz-O-Mix 11 andEnz-O-Mix 12 are designed to convert fragmented DNA into a WholeMethylome Library that can be amplified after or during the WholeMethylome Library synthesis by transcription and strand displacement,respectively.

FIG. 19E shows schematically compositions of Enz-O-Mix reagents 13 and14 designed to convert fragmented and HMW DNA, respectively, into aWhole Genome Library that can be directly cloned into an appropriatevector.

FIG. 20 illustrates assembly, storage and use of Enz-O-Mix reagent(s) asa kit in high throughput applications and clinical diagnostics, forexample.

FIG. 21A demonstrates the optimization process for Enz-O-Mix UniversalBuffer using real-time PCR to monitor amplification of the generatedlibrary.

FIG. 21B demonstrates the optimization process for Enz-O-Mix incubationtemperature using real-time PCR to monitor amplification of thegenerated library.

FIG. 21C demonstrates the optimization process for Enz-O-Mix reagent (N)concentration using real-time PCR to monitor amplification of thegenerated library.

FIG. 22 shows real-time amplification curves of whole genome librariesprepared in a single step by ligation of a stem-loop oligonucleotidewith EcoNI restriction site generated after ligation andstrand-displacement extension of blunt-end AluI restriction fragments inthe presence or in the absence of EcoNI. Excision of the terminalinverted repeat by EcoNI results in high amplification efficiency thatexceeds amplification efficiency of the library with inverted repeats(in the absence of the Eco NI cleavage) by two orders of magnitude.

FIG. 23 shows real-time amplification curves of libraries prepared in asingle step by ligation of blunt-end AluI restriction fragments to thestem-loop oligonucleotide comprising hexa-ethylene glycol replicationstop (HEG Stem-Loop Oligo), a stretch of 5 ribonucleosides (RiboStem-Loop Oligo), or just deoxyribonucleosides (Stem-Loop Oligo) intheir loop regions. Unlike the libraries made with HEG-containingoligonucleotide, the libraries with ribonucleosides in the loop regionare not amplified efficiently, suggesting that ribonucleosides in thetemplate strand do not arrest DNA replication carried by the Klenowfragment of DNA polymerase I.

FIGS. 24A, 24B, and 24C show the real-time PCR amplification curves of 3exemplary human STS markers used for evaluation of genomic sequencerepresentation in WGA libraries prepared in a single step by ligation ofblunt-end AluI restriction fragments to a stem-loop oligonucleotidecontaining within a loop either the hexa-ethylene glycol linker (HEG) asa replication stop, or the EcoNI recognition sequence that becomesfunctional (cleavable) after ligation and strand-displacement extension.Twenty nanograms of purified material of each library is compared to 1ng of human genomic DNA randomly fragmented to an average size of 1.5 Kbusing Hydro Shear device (gDNA). In FIG. 24A, there is PCR using STS #4primers. In FIG. 24B, there is PCR using STS #19 primers. In FIG. 24C,there is PCR using STS #35 primers (Table II). Results show little or norepresentational bias for all 3 STS sequences.

FIG. 25 shows the real-time amplification curves of libraries preparedin a single step by ligation of blunt-end AluI restriction fragments tothe stem-loop oligonucleotide containing deoxy-uridine in the 5′ stemregion and in the loop with T4 DNA ligase and Klenow fragment of DNApolymerase I, in the presence or in the absence Uracil-DNA glycosylase(which is also referred to as UDG or dU glycosylase). Amplification isconducted by either adding universal primer M_(U) or using the primerreleased as a result of the UDG activity, followed by heat-induceddegradation of the stem-loop oligonucleotide.

FIG. 26 illustrates a one-step genomic DNA restriction digestion andwhole genome amplification using degradable stem-loop oligonucleotidecontaining deoxy-uridine in the 5′ stem region and in the loop. Humangenomic DNA in amounts ranging from 20 ng to 10 pg or a blank sample (noDNA control) are incubated with the stem-loop oligonucleotide in thepresence of 4 exemplary enzymatic activities: AluI restrictionendonuclease, T4 DNA ligase, Klenow fragment of DNA polymerase I, andUracil-DNA glycosylase (UDG) for 1 hour at 37° C. and amplified by PCRusing universal primer M_(U).

FIG. 27 shows PCR amplification curves of specific promoter sites fromamplified libraries prepared from non-methylated (N) and artificiallymethylated (M) cell-free urine DNA from a healthy donor. Libraries wereprepared by ligation of a degradable stem-loop oligonucleotidecontaining deoxy-uridine with (N+, M+) or without (N−, M−) simultaneouscleavage with methylation-sensitive restriction enzymes. Promoter sitesfrom non-methylated cleaved DNA (N+) amplify with significant (at least10 cycles) delay as compared to uncut DNA (N−, M−) unlike methylated DNAwhich is refractory to cleavage (M+ versus M−).

FIG. 28 shows the products of simultaneous whole bacteriophage DNAlibrary synthesis and isothermal amplification by T7 polymerasetranscription of lambda DNA digested with restriction endonuclease BstEII on an agarose gel in the presence (+T4 ligase) or absence of DNAligase (−T4 ligase).

FIG. 29 shows the products of simultaneous library synthesis andisothermal amplification of cell-free urine DNA using the nickingactivity of the nuclease N.BbvC IB and strand displacement DNAsynthesis.

FIGS. 30A-30B compare two Enz-O-Mix library synthesis/PCR amplificationprocesses (Enz-O-Mix 1 and Enz-O-Mix 4 from FIGS. 19A and 19B). FIG. 30Ashows a 2-step, opened-tube protocol. In this case, Enz-O-Mix librarysynthesis/amplification is performed in two operational steps: step 1—atube containing DNA is supplemented with the library (WGA or WMA)synthesis reagents and incubated at 37° C. for 1 h, and step 2—the tubeis opened, supplemented with PCR amplification buffer/reagents andsubjected to temperature cycling. FIG. 30B shows a 1-step, closed-tubeprotocol. In this case, Enz-O-Mix library synthesis/amplification isperformed in one operational step when a tube containing DNA issupplemented with the library synthesis/amplification (PCR) reagents anduniversal buffer, and subjected to programmed temperature conditionswithin a thermocycler.

FIG. 31 shows an expected accumulation of DNA in a tube (a) and atypical temperature profile (b) for a one-step, closed tube Enz-O-MixPCR-based WGA or WMA.

FIG. 32 illustrates heat-induced conversion of a stem-loopoligonucleotide adaptor containing dU-bases within the loop and the 5′stem region into a functional universal PCR primer.

FIG. 33 shows effect of DMSO and magnesium concentration on the wholegenome amplification of HMW human DNA isolated from blood usingintegrated one-step, closed-tube Enz-O-Mix protocol illustrated in FIGS.30, 31, and 32, and real-time PCR detection.

FIG. 34 illustrate application of the one-step, closed tube Enz-O-MixWGA process (DNA fragmentation, amplification, and labeling) formicro-array analysis.

FIGS. 35A and 35B show the one-step process for simultaneous GenomePlexor MethylPlex library synthesis and immobilization. In FIG. 35A, thereis non-covalent library immobilization by hybridization of the exposed5′ stem region to an oligo S covalently attached to a tube/well surface.In FIG. 35B, there is a covalent library immobilization by hybridizationand ligation of the exposed 5′ stem region to an oligo S covalentlyattached to a tube/well surface.

FIG. 36 shows tubes, plates, micro-slides, and micro-beads manufacturedfor one-step GenomePlex or MethylPlex library synthesis andimmobilization.

FIG. 37 shows a hypothetical fluidic device that utilizes the one-stepGenomePlex or MethylPlex library synthesis and immobilization process.

FIG. 38 illustrates hot start locus-specific PCR amplification of DNAusing degradable stem-loop primers.

FIG. 39 shows the specific events that lead to a conversion of theinactive stem-loop oligonucleotides into the active PCR primers.

FIG. 40 shows accumulation of a specific PCR product (A) and a typicaltemperature profile (B) for a hot start PCR using degradable stem-loopprimers

DETAILED DESCRIPTION OF THE INVENTION

The present application incorporates by reference herein in its entiretyU.S. patent application Ser. No. 11/071,864, filed Mar. 3, 2005. Alsoincorporated by reference herein in its entirety is U.S. patentapplication Serial No. unknown, filed Mar. 2, 2006, entitled “Isolationof CpG Islands by Thermal Segregation and EnzymaticSelection-Amplification Method,” which itself claims priority to U.S.Provisional Patent Application Ser. No. 60/704,541, filed Aug. 2, 2005.

I. Definitions

In keeping with long-standing patent law convention, the words “a” and“an” when used in the present specification in concert with the wordcomprising, including the claims, denote “one or more.” Some embodimentsof the invention may consist of or consist essentially of one or moreelements, method steps, and/or methods of the invention. It iscontemplated that any method or composition described herein can beimplemented with respect to any other method or composition describedherein.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiology,recombinant DNA, and so forth which are within the skill of the art.Such techniques are explained fully in the literature. See e.g.,Sambrook, Fritsch, and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL,Second Edition (1989), OLIGONUCLEOTIDE SYNTHESIS (M. J. Gait Ed., 1984),ANIMAL CELL CULTURE (R. I. Freshney, Ed., 1987), the series METHODS INENZYMOLOGY (Academic Press, Inc.); GENE TRANSFER VECTORS FOR MAMMALIANCELLS (J. M. Miller and M. P. Calos eds. 1987), HANDBOOK OF EXPERIMENTALIMMUNOLOGY, (D. M. Weir and C. C. Blackwell, Eds.), CURRENT PROTOCOLS INMOLECULAR BIOLOGY (F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore,J. G. Siedman, J. A. Smith, and K. Struhl, eds., 1987), CURRENTPROTOCOLS IN IMMUNOLOGY (J. E. Coligan, A. M. Kruisbeek, D. H.Margulies, E. M. Shevach and W. Strober, eds., 1991); ANNUAL REVIEW OFIMMUNOLOGY; as well as monographs in journals such as ADVANCES INIMMUNOLOGY. All patents, patent applications, and publications mentionedherein, both supra and infra, are hereby incorporated herein byreference.

A skilled artisan recognizes that there is a conventional single lettercode in the art to represent a selection of nucleotides for a particularnucleotide site. For example, R refers to A or G; Y refers to C or T; Mrefers to A or C; K refers to G or T; S refers to C or G; W refers to Aor T; H refers to A or C or T; B refers to C or G or T; V refers to A orC or G; D refers to A or G or T; and N refers to A or C or G or T.

The term “blunt end” as used herein refers to the end of a dsDNAmolecule having 5′ and 3′ ends, wherein the 5′ and 3′ ends terminate atthe same nucleotide position. Thus, the blunt end comprises no 5′ or 3′overhang.

The term “double stranded molecule” as used herein refers to a moleculethat is double stranded at least in part.

The term “homing endonuclease” as used herein refers to proteins thatare encoded by polynucleotides having mobile, self-splicing introns.Homing endonucleases promote the movement of at least part of the DNAsequences that encode them from one polynucleotide location to anotherby generating a site-specific double-stranded break at a target site inan allele that lacks the corresponding mobile intron. Examples includebut are not limited to at least the following: I-Ceu I, I-Sce I, Pl-PspI, Pl-Sce I, or a mixture thereof.

The terms “hairpin” and “stem-loop oligonucleotide” as used herein referto a structure formed by an oligonucleotide comprised of 5′ and 3′terminal regions that are inverted repeats and a non-self-complementarycentral region, wherein the self-complementary inverted repeats form adouble-stranded stem and the non-self-complementary central region formsa single-stranded loop.

The term “in the absence of exogenous manipulation” as used hereinrefers to there being modification of a DNA molecule without changingthe solution in which the DNA molecule is being modified. In specificembodiments, it occurs in the absence of the hand of man or in theabsence of a machine that changes solution conditions, which may also bereferred to as buffer conditions. In further specific embodiments,changes in temperature occur during the modification.

The term “nonidentical function” as used herein refers to two or moreenzymes that do not comprise the same activity. For example, tworestriction endonucleases would be considered to have identicalfunction, although a restriction endonuclease and a ligase would beconsidered to have nonidentical function. Further, endonucleases aredefined as enzymes that cut double stranded DNA and therefore that haveidentical function, although this may be performed in a variety of ways.For example, some restriction endonucleases cut frequently enough thatthey may be considered to have a DNA fragmentation function (such as toreduce an average DNA size to the size appropriate for efficient WGA,for example), and other restriction endonucleases have a function ofcleaving unmethylated restriction sites, for example to generate aMethylome library (such as cleavage that would not reduce an average DNAsize), for example.

The term “polished” as used herein refers to the repair of dsDNAfragment termini that may be enzymatically repaired, wherein the repairconstitutes the fill in of recessed 3′ ends or the exonuclease activitytrimming back of 5′ ends to form a “blunt end” compatible with adaptorligation.

II. Specific Embodiments of the Invention

The present invention may employ particular compositions and methods asdescribed in the following exemplary embodiments.

A. One-Step Attachment of Double-Stranded Inverted Repeat DNA Sequencesto DNA Fragments Using Stem-Loop Oligonucleotides

In this embodiment of the present invention, as illustrated in FIG. 3,DNA is incubated with an exemplary mixture comprising a stem-loopoligonucleotide with 3′ recessed, 3′ protruding, or blunt end (FIG. 4);a 3′ proofreading DNA polymerase (Klenow fragment of the DNA polymeraseI, T4 DNA polymerase, etc.); T4 DNA ligase; Enz-O-Mix Universal Buffer;ATP; and dNTPs. Four exemplary enzymatic reactions are taking placesimultaneously: “polishing” of the DNA ends and the oligonucleotidedouble-stranded stem-region; ligation of the oligonucleotide 3′ end tothe 5′ phosphate of the DNA leaving a nick between the 3′ end of DNA andthe 5′ end of the oligonucleotide double-stranded stem-region;polymerase extension of the 3′ DNA end that propagates toward the end ofthe stem-loop oligonucleotide; and a strand-displacement reaction withinthe oligonucleotide stem region. The process results in a library of DNAfragments with inverted repeat adaptors at their ends.

B. Transcription-Mediated Amplification of DNA Library with AttachedPromoter Sequence

In this embodiment of the present invention, as illustrated in FIG. 5, aDNA library produced by incubation with an exemplary mixture comprisinga stem-loop oligonucleotide with 3′ recessed, 3′ protruding, or bluntend (FIG. 4) and T7 promoter sequence within the stem or loop region; a3′ proofreading DNA polymerase (Klenow fragment of the DNA polymerase I,T4 DNA polymerase, etc.); T4 DNA ligase; Enz-O-Mix Universal Buffer;ATP; and dNTPs (as shown in FIG. 3), is used as a template for thetranscription-mediated amplification. In FIG. 5A, promoter sequenceresides within the stem region of a stem-loop oligonucleotide, and thereare two inversely oriented promoters at the ends of all DNA fragments:internally located promoter that initiates synthesis of long RNAmolecules, and terminal promoter that initiates synthesis of very short(<10 bases) RNA initiation products (not shown in FIG. 5A) (similarproducts will be produced from the non-attached stem-loopoligonucleotides present in the mixture). In FIG. 5B, the promotersequence resides within the loop region of a stem-loop oligonucleotide,and there is only one active promoter element that supports synthesis oflong RNA products. The transcription-mediated amplification is achievedby incubation of the synthesized DNA library (see above) with T7 RNApolymerase in the presence of ribonucleotides (rNTPs). The products ofthis reaction are RNA molecules.

Supplementation of a transcription-mediated amplification reaction withadditional ingredients, such as a reverse transcriptase polymerase(M-MuLV reverse transcriptase, AMV reverse transcriptase, etc.) and aprimer complementary to the T7 promoter or the internal stem region atthe 3′ end of the RNA molecule (see FIG. 6B), would allow the synthesisof a complementary cDNA. Efficient priming at the 3′ ends of RNA isachieved by blocking the transcription-mediated RNA synthesis within theadaptor region to prevent formation of a folded structure at the 3′ endsof RNA. Transcriptional arrest can be achieved by using the samechemical or enzymatic modifications of a stem-loop oligonucleotide asdescribed herein in Sections E, F, G, and H and shown in FIGS. 8, 10,and 16C. The products of such a reaction are double stranded DNA/RNAhybrid molecules.

C. Enz-O-Mix Reagents and Procedures for One-Step Library Synthesis andLinear Amplification by Transcription

This embodiment is shown in FIG. 6A and illustrates exemplary componentsand enzymatic reactions involved in the process catalyzed by Enz-O-Mixreagent (Enz-O-Mix 5, FIG. 19B). This reagent simultaneously synthesizesa DNA library with T7 promoter at the ends and amplifies it linearly bytranscription. The Enz-O-Mix reagent 5 comprises a stem-loopoligonucleotide with T7 promoter sequence in the loop region; a DNAfragmentation endonuclease (a restriction enzyme, DNase I,methylation-specific nuclease McrBC, benzonase, apoptotic endonuclease,etc.); a 3′ proofreading DNA polymerase; T4 DNA ligase; T7 RNApolymerase; Enz-O-Mix Universal Buffer; rNTP; and dNTPs. First, HMW DNAis fragmented by an endonuclease to produce 100-3,000 bp DNA fragments.The T4 DNA polymerase “polishes” the DNA ends and the stem-loopoligonucleotide double-stranded stem-region, and T4 ligase ligates the3′ end of the oligonucleotide to the 5′ phosphate of the DNA leaving anick between the 3′ end of DNA and the 5′ end of the oligonucleotidedouble-stranded stem-region. DNA polymerase extends the available 3′ DNAend toward the end of a stem-loop oligonucleotide and generates alibrary of DNA fragments with active conformation of T7 promoter attheir ends. RNA polymerase transcribes the library, linearly amplifyingDNA and producing single stranded RNA molecules.

In the case of fragmented DNA (for example, cell-free DNA from bloodand/or urine) the reaction does not require a fragmentation endonuclease(Enz-O-Mix 2, FIG. 19A); all other components may be the same asdescribed above.

In another embodiment, as shown in FIG. 6B, supplementation of atranscription-mediated amplification reaction with the additionalingredients, such as a reverse transcriptase polymerase (M-MuLV reversetranscriptase, AMV reverse transcriptase, etc.) and T7 primer, allowsthe synthesis of a complementary cDNA. Efficient priming at the 3′ endsof RNA is achieved by blocking the transcription-mediated RNA synthesiswithin the adaptor region to prevent formation of a folded structure atthe 3′ ends of RNA. Transcriptional arrest can be achieved by using thesame chemical or enzymatic modifications of a stem-loop oligonucleotideas described herein in Sections E, F, G, and H and shown in FIGS. 8, 10,and 16C. The products of such a reaction are double stranded DNA/RNAhybrid molecules.

D. Inhibitory Effect of Inverted Repeats at DNA Ends on Whole GenomeAmplification and Locus-Specific PCR

As shown in FIG. 7, inverted repeats attached to both ends of DNAfragments have an inhibitory effect on PCR process. Heating to 95° C.during a first PCR cycle denatures DNA strands and generateshairpin-like structures at both 3′ and 5′ ends of DNA fragments due tothe folding of terminal inverted repeat sequences (the meltingtemperature for these structures is usually more than 100° C.).Subsequent cooling activates a thermostable DNA polymerase and resultsin extension of the self-primed 3′ ends and creation of long hairpin DNAmolecules refractory to melting, priming, and PCR amplification. Neitherthe universal primer A nor any internal, site-specific primer pair wouldproduce any detectable PCR product.

E. Library Created by Enz-O-Mix Attachment Process with a Stem-LoopOligonucleotide with a Non-Replicable Linker

This embodiment is illustrated in FIG. 8 and describes the one-stepEnz-O-Mix attachment process for a stem-loop oligonucleotide with anon-replicable linker. The exemplary reaction mix comprises fragmentedDNA; a stem-loop oligonucleotide with 3′ recessed, 3′ protruding orblunt end (FIG. 4), and a non-replicable linker (such as about in thecentral part of the oligonucleotide); a 3′ proofreading DNA polymerase(Klenow fragment of the DNA polymerase I, T4 DNA polymerase, etc.); T4DNA ligase; Enz-O-Mix Universal Buffer; ATP; and dNTPs. At least thefollowing enzymatic reactions are taking place: “polishing” of the DNAends and the stem-loop oligonucleotide double-stranded stem-region;ligation of the oligonucleotide 3′ end to the 5′ phosphate of the DNA,leaving a nick between the 3′ end of DNA and the 5′ end of theoligonucleotide double-stranded stem-region; polymerase extension of the3′ DNA end that propagates toward the end of stem-loop oligonucleotideand stops somewhere within the loop or close to the loop region at thereplication block, for example. The process results in the library ofDNA fragments with universal sequence A at the ends and an invertedrepeat attached only to the 5′ end of DNA.

F. Libraries Produced by the Enz-O-Mix Attachment Process with aStem-Loop Oligonucleotide Comprising a Non-Replicable Linker have NoInhibitory Effect on PCR

As shown in FIG. 9, a DNA library produced by the Enz-O-Mix (Enz-O-Mix 1in the case of fragmented DNA, FIG. 19A, or Enz-O-Mix 4 in the case ofHMW DNA, FIG. 19B) attachment process with a stem-loop oligonucleotidewith a non-replicable linker has no inhibitory effect on WGA and PCR.Heating to 95° C. during first PCR cycle denatures DNA strands andgenerates hairpin-like structures only at the 5′ ends of DNA fragments.At a lower PCR temperature, the universal primer A anneals to the A′region at the 3′ ends of DNA fragments, and a thermostable DNApolymerase replicates DNA until it reaches a non-replicable linker inthe loop or in the region adjacent to the loop stem region of thestem-loop oligonucleotide attached to the 5′ DNA end. Replication of thehairpin region is accompanied by a strand-displacement reaction withinthe oligonucleotide stem region. Synthesized DNA molecules with theuniversal sequence A and no inverted repeats at the ends (shown bydashed lines) can then successfully be amplified by PCR.

G. Stem-Loop Oligonucleotide with a Non-Replicable Linker IntroducedDuring Chemical Synthesis

FIG. 10A shows the structure of a stem-loop oligonucleotide with anon-replicable linker introduced chemically during oligonucleotidesynthesis and shows detailed events occurring at DNA end during theexemplary multi-enzyme attachment process. Many structural modificationswithin the oligonucleotide, such as the exemplary hexaethelene glycollinker, the abasic site, or a cluster of abasic sites (a gap), a bulkygroup within the base or backbone, etc. can block propagation of the DNAreplication. The exemplary process occurs as described in FIG. 8.

H. Stem-Loop Oligonucleotide with a Non-Replicable Abasic Gap GeneratedEnzymatically by dU-Glycosylase During the Attachment Process

This embodiment is illustrated in FIG. 10B and shows the structure of astem-loop oligonucleotide with non-replicable linker introducedenzymatically during the attachment reaction and shows detailed eventsoccurring at a DNA end during the exemplary multi-enzyme attachmentprocess. The exemplary reaction mix comprises fragmented DNA; astem-loop oligonucleotide with 3′ recessed, 3′ protruding or blunt end(FIG. 4), and dU bases within the loop and stem regions (optional); adU-glycosylase; a 3′ proofreading DNA polymerase; T4 DNA ligase;Enz-O-Mix Universal Buffer; ATP; and dNTPs. dU-glycosylase createsabasic sites within the loop and upper strand of the stem region of theoligonucleotide; DNA polymerase “polishes” the ends of DNA and thestem-loop oligonucleotide and generates blunt ends; T4 DNA ligaseattaches the 3′ end of the oligonucleotide and the 5′ end of DNAfragment, leaving a nick between the 3′ end of DNA and the 5′ end of thehairpin double-stranded stem-region; a DNA polymerase extends the 3′ endof DNA toward the end of the stem-loop oligonucleotide and stopssomewhere within the loop or close to the loop region of theoligonucleotide, such as at the replication block generated by acontiguous abasic site region. The role of abasic sites in the aboutcentral stem region is to destabilize base pairing and facilitate thestrand displacement reaction. The process results in a library of DNAfragments with universal sequence at both ends and an inverted repeatattached only to the 5′ end of DNA, thereby suitable for PCRamplification. Heating at 95° C. during PCR generates breaks at abasicsites and completely eliminates the 5′ portion of a stem-loopoligonucleotide.

I. Stem-Loop Oligonucleotides are Advantageous for the Enz-O-MixAttachment Process

This embodiment illustrates the low efficiency and problems associatedwith use of standard adaptors in the Enz-O-Mix attachment process,although in alternative embodiments standard adaptors may be employed.

FIG. 11A shows a standard adaptor formed by two differentoligonucleotides, wherein one of them has the 5′ phosphate group, andpossible products formed when such an adaptor is used in themulti-enzyme one-step ligation process. The adaptor should be present athigh concentration to provide efficient ligation to DNA ends. Becauseboth ends of a standard adaptor can be ligated to DNA and formadaptor-adaptor conjugates (a dominating product of the reaction), anumber of undesirable products may be high, and the yield of desirablemolecules may be low, in certain aspects.

FIG. 11B shows a standard adaptor formed by two differentoligonucleotides without phosphate group and two products formed whensuch an adaptor is used in the multi-enzyme one-step ligation process.Only 50% of formed molecules are desirable products with correctorientation of the adaptor, while the other 50% of molecules haveinversed adaptor orientation.

FIG. 11C shows a standard adaptor formed by two differentoligonucleotides without phosphate group but with a protective group atthe 3′ end of one oligonucleotide and the products formed when suchadaptor is used in the multi-enzyme one-step ligation process. A blocked3′ end or recessive 3′ end or both are well-known in the art and havebeen used to prevent formation of products with inversed adaptororientation during ligation reaction. Presence of a 3′ proofreading DNApolymerase in the mix will replace the terminal 3′ blocked nucleotidewith normal nucleotide (a), and repair and extend the recessed, blocked3′ end (b). As in the case described for FIG. 11B, only 50% of formedmolecules are desirable products with correct orientation of theadaptor, while the other 50% of molecules have inversed adaptororientation.

J. Library Generated by the Enz-O-Mix Attachment Process Using aStem-Loop Oligonucleotide with a Cleavable Site Generated During theMulti-Enzyme Reaction

In this embodiment of the present invention, as illustrated in FIG. 12,there is a schematic description of the one-step Enz-O-Mix attachmentprocess for a stem-loop oligonucleotide with an enzymatically cleavablesite generated during the multi-enzyme reaction. The reaction mixcontains fragmented DNA, a stem-loop oligonucleotide with 3′ recessed,3′ protruding or blunt end (FIG. 4), and a specific DNA sequence Rwithin the loop or in the loop and adjacent stem region; a 3′proofreading DNA polymerase (Klenow fragment of the DNA polymerase I, T4DNA polymerase, etc.); T4 DNA ligase; an endonuclease that recognizesand cuts the oligonucleotide specific oligo-sequence R when it adopts adouble-stranded conformation; Enz-O-Mix Universal Buffer; ATP; anddNTPs. Four enzymatic reactions are taking place nearly simultaneously:“polishing” of the DNA ends and the stem-loop oligonucleotidedouble-stranded stem-region; ligation of the oligonucleotide 3′ end tothe 5′ phosphate of the DNA leaving a nick between the 3′ end of DNA andthe 5′ end of the hairpin double-stranded stem-region, polymeraseextension of the 3′ DNA end that propagates toward the end of stem-loopoligonucleotide and generates inverted repeats at the ends of DNAfragments; and, finally, cleavage of inverted repeats by an endonucleaseat the R sites. The process results in a library of DNA fragments withuniversal sequence A at the ends, and no inverted repeat attached to theends of DNA fragments, which is thereby competent for PCR amplification.

K. Examples of Stem-Loop Adaptors with a Cleavable Restriction SiteGenerated by DNA Synthesis

FIG. 13A shows an exemplary stem-loop oligonucleotide with recognitionsequence for the restriction endonuclease Eco NI located at the loopregion. The enzyme can not cut the CCTNNNNNAGG (SEQ ID NO: 20) regionwithin the stem-loop oligonucleotide (an unrecognizable structure) butit will cut it efficiently when the oligonucleotide is attached to a DNAend and adopts a canonical (double-stranded) Watson-Crick conformation.Cleavage with Eco NI eliminates inverted repeats that inhibit PCRapplications.

FIG. 13B shows an exemplary stem-loop oligonucleotide with recognitionsequence for the restriction endonuclease Pac I located within the loopregion. The enzyme can not cut the TTAATTAA region within the stem-loopoligonucleotide, but it will cut it efficiently when the oligonucleotideis attached to a DNA end and adopts a canonical (double-stranded)Watson-Crick conformation. Cleavage eliminates inverted repeats thatinhibit PCR applications. Use of a rare-cutting restriction enzyme suchas Pac I reduces the number of DNA fragments affected by the enzymecleavage.

FIG. 13C shows the stem-loop oligonucleotide with recognition sequencefor the homing endonuclease I-Ceu I located within the loop region. Theenzyme can not cut the 26-base single-stranded region within the loop,but it cuts efficiently when the stem-loop oligonucleotide is attachedto a DNA end and adopts a canonical (double-stranded) Watson-Crickconformation. Cleavage eliminates inverted repeats that inhibit PCRapplications. Use of a homing endonuclease dramatically reduces andpractically eliminates cleavage of genomic DNA.

L. Library Generated by the Enz-O-Mix Attachment Process with aStem-Loop Oligonucleotide with a Short Stem

Amplifiable libraries can be synthesized in the presence of but notlimited to a stem-loop oligonucleotide with a non-replicable orcleavable linker. FIG. 14 shows a schematic description of an exemplaryone-step Enz-O-Mix attachment process using a stem-loop oligonucleotidewith a very short stem. The exemplary reaction mix comprises fragmentedDNA, a stem-loop oligonucleotide with 3′ recessed, 3′ protruding orblunt end (FIG. 4), and a short stem region (5-8 bases); a 3′proofreading DNA polymerase (Klenow fragment of the DNA polymerase I, T4DNA polymerase, etc.); T4 DNA ligase; Enz-O-Mix Universal Buffer; ATP;and dNTPs. Three enzymatic reactions are taking place: “polishing” ofthe DNA ends and the stem-loop oligonucleotide double-strandedstem-region; ligation of the oligonucleotide 3′ end to the 5′ phosphateof the DNA, leaving a nick between the 3′ end of DNA and the 5′ end ofthe hairpin double-stranded stem-region; and polymerase extension of the3′ DNA end that propagates toward the end of the stem-loopoligonucleotide and generates short inverted repeats at the ends of DNAfragments. The process results in a library of DNA fragments withuniversal sequence A at the ends and a very short inverted repeatattached to the ends of DNA fragments (FIG. 14, shown in bold). PCR ispossible because the melting temperature for terminal hairpins is lowenough to prevent folding, self-priming and formation of long hairpinmolecules, as shown on FIG. 7. Primer A (representing the 3′ portion ofa stem-loop oligonucleotide without the complementary 5′ region) or eventhe whole stem-loop oligonucleotide can be utilized as a primer for PCRamplification.

M. Enz-O-Mix Reagents and Procedures for One-Step Library Synthesis andOne-Step Library Synthesis/Amplification Utilizing Strand DisplacementDNA Amplification

This embodiment is shown in FIG. 15 and illustrates exemplary componentsand enzymatic reactions involved in the process catalyzed by Enz-O-Mixreagents (Enz-O-Mix 3 and Enz-O-Mix 6, FIGS. 19A and 19B). The reagentEnz-O-Mix 3 can use fragmented (cell-free serum DNA or urine DNA, forexample), while the reagent Enz-O-Mix 6 can utilize HMW DNA andsynthesize a library of DNA fragments with the recognition sites for anicking endonuclease (N.AlwI, N.BbvCIA, N.BbvCIB, Nb.Bpu10I, Nb.BsmI,N.Bst9I, N.BstNBI, etc.) at their ends to allow their subsequentamplification by strand displacement DNA synthesis. The presence ofnicking endonuclease and strand displacing polymerase in the reagentsEnz-O-Mix 3 and Enx-O-Mix 6 allows one to simultaneously synthesize andamplify DNA library by strand displacement DNA synthesis.

For example, the Enz-O-Mix reagent 3 (suitable for fragmented DNA)comprises a stem-loop oligonucleotide with a recognition sequence for anicking nuclease; a primer P complementary at least partially to thestem region of a stem-loop oligonucleotide; a 3′ proofreading DNApolymerase; a strand displacing DNA polymerase; T4 DNA ligase; a nickingendonuclease; Enz-O-Mix Universal Buffer; and dNTPs. T4 DNA polymerase“polishes” the DNA ends and the stem-loop oligonucleotidedouble-stranded stem-region. T4 ligase ligates the 3′ end of theoligonucleotide to the 5′ phosphate of the DNA, leaving a nick betweenthe 3′ end of DNA and the 5′ end of the oligonucleotide double-strandedstem-region. DNA polymerase extends the available 3′ DNA end toward theend of a stem-loop oligonucleotide and generates a library of DNAfragments with functional recognition site for a nicking endonuclease(N.AlwI, N.BbvCIA, N.BbvCIB, Nb.Bpu10I, Nb.BsmI, N.Bst9I, N.BstNBI,etc.) at their ends. DNA polymerase initiates multiple rounds of stranddisplacement DNA synthesis at nicks generated by a nicking nuclease,followed by synthesis of a second DNA strand (primed by primer P),producing double stranded DNA molecules and linearly amplifying DNA.

In the case of HMW DNA (Enz-O-Mix 6, FIG. 19B), the reaction requires aDNA fragmentation endonuclease (a restriction enzyme, DNase I,methylation-specific nuclease McrBC, benzonase, apoptotic endonucleaseetc.), and all other components may be the same as described above forEnz-O-Mix 3.

N. Examples of Stem-Loop Adaptors with a Nicking EndonucleaseRecognition Site Generated by DNA Synthesis

FIG. 16A shows the stem-loop oligonucleotide with recognition sequencefor the exemplary nicking endonuclease N.Bbv CIA located at the loopregion. The enzyme can not nick the single stranded GCTGAGG regionwithin the stem-loop oligonucleotide (an unrecognizable structure), butit will nick it efficiently when the oligonucleotide is attached to aDNA end and adopts a canonical (double-stranded) Watson-Crickconformation.

FIG. 16B shows an exemplary stem-loop oligonucleotide with recognitionsequence for the nicking endonuclease N.Bbv CIA located at the end ofthe stem region. The enzyme can not nick efficiently the terminallylocated TTAATTAA region within the stem-loop oligonucleotide, but itwill nick it efficiently when the oligonucleotide is attached to a DNAend and the site acquires an internal location. A second nicking site,generated by the oligonucleotide attachment process, would have aterminal location and low nicking efficiency.

FIG. 16C shows an exemplary stem-loop oligonucleotides 1 and 2 thatcontains several functional elements within the loop region includingthe recognition sequence for a nicking endonuclease. Multifunctionalstem-loop oligonucleotide have several regulatory elements within theloop region: T7 promoter sequence (that includes all regions necessaryfor transcription); recognition sequence N for a nicking endonuclease(N.AlwI, N.BbvCIA, N.BbvCIB, Nb.Bpu10I, Nb.BsmI, N.Bst9I, N.BstNBI,etc.); recognition sequence for a restriction endonuclease; and areplication/transcription block region (a chemical modification or astring of 2-4 dU bases). The regulatory elements can reside within asingle loop region (L1 in the stem-loop oligonucleotide 1), or bedistributed across several loop regions (L1, L2, and L3 in the stem-loopoligonucleotide 2). A multifunctional stem-loop oligonucleotide cansupport different types of DNA amplification, including PCR, isothermalamplification by transcription, isothermal amplification by stranddisplacement synthesis, or even a combination thereof and can alsofacilitate modification such as DNA immobilization, DNA recombination,DNA cloning, or a combination thereof. Activation of the RNAtranscription, DNA nicking, DNA double-stranded cleavage, and generationof the 5′ overhang functions of the stem-loop adaptor occurs only uponits enzymatic processing and attachment to DNA ends.

O. Enz-O-Mix is a General Method for Synthesis and Amplification of DNALibraries

This embodiment of the present invention, as illustrated in FIG. 17,shows generalization of the Enz-O-Mix method and demonstrates itsspecific applications. When Enz-O-Mix reagent is combined with DNA andincubated at optimal temperature (for example, 37° C.) for a timenecessary to complete the reaction (for example, 1 hour), although itcan be more or less, it results in either a DNA library that can beamplified or cloned in a separate reaction, or a DNA library that isamplified isothermally during the incubation with the Enz-O-Mix reagent.For example, the exemplary products of reaction could be as follows: 1)a library of DNA fragments with universal sequence or sequences attachedto the ends of DNA fragments so that it can be amplified by PCR (PCR WGALibrary); 2) a library of DNA fragments with a promoter sequenceattached at the ends of DNA fragments so that it can be amplified bytranscription (Transcription WGA Library); 3) a Methylome Library, i.e.the library of adapted DNA fragments where all non-methylated CpGislands are eliminated (such as are described in U.S. patent applicationSer. No. 11/071,864, filed Mar. 3, 2005, which is incorporated byreference herein in its entirety); 4) a library of DNA fragments withready-to-clone sticky-end adaptors at the ends; 5) an amplified DNAlibrary where the product is DNA or RNA; and/or 6) an amplifiedMethylome library where the product is DNA or RNA.

P. Stem-Loop Oligo Attachment Master Mix is a Key Component of allEnz-O-Mix Reactions

This embodiment is shown in FIG. 18 and illustrates the components offour different Stem-Loop Oligo Attachment Master Mixes described in theinvention and utilized in different Enz-O-Mix reactions involved in theDNA library synthesis. The Stem-Loop Oligo Attachment Master Mix is akey component of the Enz-O-Mix reaction, and it is present in allEnz-O-Mix reagents.

The exemplary Stem-Loop Oligo Attachment Master Mix I comprises astem-loop oligonucleotide; a 3′ proofreading DNA polymerase; a DNAligase; Enz-O-Mix Universal Buffer; ATP; and dNTPs (see also FIG. 3 andFIG. 14).

The exemplary Stem-Loop Oligo Attachment Master Mix II comprises astem-loop oligonucleotide with a non-replicable chemically introducedlinker; a 3′ proofreading DNA polymerase; a DNA ligase; Enz-O-MixUniversal Buffer; ATP; and dNTPs (see also FIG. 10A).

An exemplary Stem-Loop Oligo Attachment Master Mix III comprises astem-loop oligonucleotide with a non-replicable enzymatically-introducedabasic linker; dU-glycosylase; a 3′ proofreading DNA polymerase; a DNAligase; Enz-O-Mix Universal Buffer; ATP; and dNTPs (see also FIG. 10B).

An exemplary Stem-Loop Oligo Attachment Master Mix IV comprises astem-loop oligonucleotide with a cleavable site introduced by DNAreplication; a site-specific endonuclease; a 3′ proofreading DNApolymerase; a DNA ligase; Enz-O-Mix Universal Buffer; ATP; and dNTPs(see also FIG. 12 and FIGS. 14A, 14B, and 14C).

Q. Enz-O-Mix Reagents and Procedures for the One-Step Whole GenomeLibrary Synthesis and Combined Whole Genome LibrarySynthesis-Amplification

Whole Genome Amplification (WGA) is a process that is used to amplifytotal genomic DNA for many important research and diagnosticapplications when the amount of DNA is limited. WGA is frequentlypreceded by a multi-step WGA library synthesis process. Theseembodiments describe the Enz-O-Mix reagents and procedures for theone-step Whole Genome Library synthesis and combined one-step,close-tube Whole Genome Library synthesis-amplification processes.

FIG. 19A shows schematically compositions of three Enz-O-Mix reagents,specifically Enz-O-Mix 1, Enz-O-Mix 2, and Enz-O-Mix 3 designed toconvert fragmented DNA into a Whole Genome Library competent foramplification by PCR, transcription, or strand displacementamplification, respectively.

Reagent Enz-O-Mix 1 may comprise Stem-Loop Oligo Attachment Master Mixesshown in FIG. 18, and it could be also supplemented with a thermophilicDNA polymerase. The library prepared by the Enz-O-Mix 1 process can beused for PCR-mediated WGA in a open-tube (two-step process) or aclose-tube (one-step process) formats (as shown in FIG. 30).

Reagent Enz-O-Mix 2 may comprise Stem-Loop Oligo Attachment Master Mixesshown in FIG. 18 (in this case a stem-loop oligonucleotide has the T7promoter sequence in the loop or stem region in addition to othernecessary base/backbone modifications), and it could be alsosupplemented with the T7 RNA polymerase and rNTPs. The Enz-O-Mix 2 canbe used for a two-step transcription-mediated Whole Genome Amplification(when the library is prepared first, and then amplified) or for aone-step ithothermal amplification process when the Whole Genome librarysynthesis and its amplification by transcription occurs simultaneously(as shown in FIG. 6).

Reagent Enz-O-Mix 3 may comprise Stem-Loop Oligo Attachment Master Mixesshown in FIG. 18 (in this case, a stem-loop oligonucleotide has arecognition sequence for a nicking endonuclease in the loop or stemregion in addition to other necessary base/backbone modifications). Itcould be also supplemented with a nicking endonuclease (N.AlwI,N.BbvCIA, N.BbvCIB, Nb.Bpu10I, Nb.BsmI, N.Bst9I, N.BstNBI, etc.), astrand displacing DNA polymerase (Klenow exo-, Phi 29, Bst I, etc.), andprimer P (FIG. 15). The Enz-O-Mix 3 can be used for a two-step stranddisplacement-mediated Whole Genome Amplification (when the library isprepared first, and then amplified) or for a one-step ithothermalamplification process when the Whole Genome library synthesis and itsamplification by a strand displacement synthesis occurs simultaneously(as shown in FIG. 15).

FIG. 19B shows schematically compositions of three Enz-O-Mix reagents,specifically Enz-O-Mix 4, Enz-O-Mix 5, and Enz-O-Mix 6 designed toconvert high molecular DNA into a Whole Genome Library competent foramplification by PCR, transcription, or strand displacementamplification, respectively.

Reagent Enz-O-Mix 4 may comprise Stem-Loop Oligo Attachment Master Mixesshown in FIG. 18; it is also supplemented with a DNA fragmentationendonuclease such as restriction enzyme DNase I, Benzonase,methylation-specific nuclease McrBC, apoptotic endonuclease, etc.; andit could be also supplemented with a thermophilic DNA polymerase. Thelibrary prepared by the Enz-O-Mix 4 process can be used for PCR-mediatedWGA in a open-tube (two-step process) or a close-tube (one-step process)formats (as shown in FIG. 30).

Reagent Enz-O-Mix 5 may comprise Stem-Loop Oligo Attachment Master Mixesshown in FIG. 18 (in this case, a stem-loop oligonucleotide has the T7promoter sequence in the loop or stem region in addition to othernecessary base/backbone modifications), and it is also supplemented witha DNA fragmentation endonuclease such as restriction enzyme DNase I,benzonase, methylation-specific nuclease McrBC, apoptotic endonuclease,etc.; and it could be also supplemented with RNA polymerase and rNTPs.The Enz-O-Mix 5 can be used for a two-step transcription-mediated WholeGenome Amplification of HMW DNA (when the library is prepared first, andthen amplified) or for a one-step ithothermal amplification process whenthe Whole Genome library synthesis and its amplification bytranscription occurs simultaneously (as shown in FIG. 6).

Reagent Enz-O-Mix 6 may comprise Stem-Loop Oligo Attachment Master Mixesshown in FIG. 18 (in this case, a stem-loop oligonucleotide has arecognition sequence for a nicking endonuclease in the loop or stemregion in addition to other necessary base/backbone modifications), andit is supplemented with a DNA fragmentation endonuclease such asrestriction enzyme, DNase I, benzonase, methylation-specific nucleaseMcrBC, apoptotic endonuclease. It could also be supplemented with anicking endonuclease (N.AlwI, N.BbvCIA, N.BbvCIB, Nb.Bpu10I, Nb.BsmI,N.Bst9I, N.BstNBI, etc.); a strand displacing DNA polymerase (Klenowexo-, Phi 29, Bst I, etc.); and primer P (FIG. 15). The Enz-O-Mix 6 canbe used for a two-step strand displacement-mediated Whole GenomeAmplification of HMW DNA (when the library is prepared first, and thenamplified) or for a one-step ithothermal amplification process when theWhole Genome library synthesis and its amplification by a stranddisplacement synthesis occurs simultaneously (as shown in FIG. 15).

R. Enz-O-Mix Reagents and Procedures for One-Step Whole MethylomeLibrary Synthesis and Combined Whole Methylome LibrarySynthesis-Amplification

Whole Methylome Amplification (WMA) is a process that was recentlydeveloped to amplify methylated DNA and suppress amplification ofnon-methylated genomic (promoter) regions for many important researchand diagnostic applications, especially when the amount of DNA islimited (see, for example, U.S. patent application Ser. No. 11/071,864,filed Mar. 3, 2005, which is incorporated by reference herein in itsentirety). In a standard protocol, WMA is preceded by a multi-step WMAlibrary synthesis process. This embodiment describes the exemplaryEnz-O-Mix reagents and procedures for the one-step Whole MethylomeLibrary synthesis and combined Whole Methylome Librarysynthesis-amplification processes.

FIG. 19C shows schematically compositions of three Enz-O-Mix reagents,Enx-O-Mix 7,Enx-O-Mix 8, and Enx-O-Mix 9, designed to convert fragmentedDNA into a Whole Methylome Library competent for amplification by PCR,transcription, or strand displacement, for example.

Reagent Enz-O-Mix 7 may comprise one of Stem-Loop Oligo AttachmentMaster Mixes shown in FIG. 18, and it is also supplemented with amixture of several (about 5-about 12) methylation-sensitive restrictionenzymes, such as Aci I, Acc II, Asp LE I, Ava I, Bce AI, Bsa HI, Bsh1236 I, Bsi E1, Bsi SI, Bst FN I, Bst HH I, Bst UI, Cfo I, Hap II, HgaI, Hha I, HinP1 I, Hin 6I, Hpa II, Hpy 99I, Hpy CH4 IV, Hsp AI, Mvn I,and Ssi I, to produce a substantial fragmentation of all non-methylatedCpG islands (see, for example, U.S. patent application Ser. No.11/071,864, filed Mar. 3, 2005, which is incorporated by referenceherein in its entirety). Reagent Enz-O-Mix 7 could be also supplementedwith a thermophilic DNA polymerase. The Methylome library prepared bythe Enz-O-Mix 7 process can be used for PCR-mediated WMA in a open-tube(two-step process) or a close-tube (one-step process) formats (as shownin FIG. 30).

Reagent Enz-O-Mix 8 may comprise Stem-Loop Oligo Attachment Master Mixesshown in FIG. 18 (in this case a stem-loop oligonucleotide has the T7promoter sequence in the loop or stem region in addition to othernecessary base/backbone modifications), and it is also supplemented witha mixture of several (about 5-about 12) methylation-sensitiverestriction enzymes, such as Aci I, Acc II, Asp LE I, Ava I, Bce AI, BsaHI, Bsh 1236 I, Bsi E1, Bsi SI, Bst FN I, Bst HH I, Bst UI, Cfo I, HapII, Hga I, Hha I, HinP1 I, Hin 6I, Hpa II, Hpy 99I, Hpy CH4 IV, Hsp AI,Mvn I, and Ssi I—to produce a substantial fragmentation of allnon-methylated CpG islands (U.S. patent application Ser. No. 11/071,864,filed Mar. 3, 2005, which is incorporated by reference herein in itsentirety). Reagent Enz-O-Mix 8 could be also supplemented with T7 RNApolymerase and rNTPs. The Enz-O-Mix 8 can be used for a two-steptranscription-mediated Whole Methylome Amplification (when the WholeMethylome library is prepared first, and then amplified) or for aone-step ithothermal amplification process when the Methylome librarysynthesis and its amplification by transcription occurs simultaneously.

Reagent Enz-O-Mix 9 may comprise Stem-Loop Oligo Attachment Master Mixesshown in FIG. 18 (in this case, a stem-loop oligonucleotide has arecognition sequence for a nicking endonuclease in the loop or stemregion in addition to other necessary base/backbone modifications), andit is also supplemented with a mixture of several (about 5-12)methylation-sensitive restriction enzymes, such as Aci I, Acc II, Asp LEI, Ava I, Bce AI, Bsa HI, Bsh 1236 I, Bsi E1, Bsi SI, Bst FN I, Bst HHI, Bst UI, Cfo I, Hap II, Hga I, Hha I, HinP1 I, Hin 6I, Hpa II, Hpy99I, Hpy CH4 IV, Hsp AI, Mvn I, and Ssi I, to produce a substantialfragmentation of all non-methylated CpG islands (see, for example, U.S.patent application Ser. No. 11/071,864, filed Mar. 3, 2005, which isincorporated by reference herein in its entirety). Reagent Enz-O-Mix 9could be also supplemented with nicking endonuclease (N.AlwI, N.BbvCIA,N.BbvCIB, Nb.Bpu10I, Nb.BsmI, N.Bst9I, N.BstNBI, etc.), a stranddisplacing DNA polymerase (Klenow exo-, Phi 29, Bst I, etc.), and primerP (FIG. 15). The Enz-O-Mix 9 can be used for a two-step stranddisplacement-mediated Whole Methylome Amplification (when the library isprepared first, and then amplified) or for a one-step ithothermalamplification process when the Methylome library synthesis and itsamplification by a strand displacement synthesis occurs simultaneously(as shown in FIG. 15).

FIG. 19D show schematically compositions of another three Enz-O-Mixreagents, Enx-O-Mix 10, Enx-O-Mix 11, and Enx-O-Mix 12, designed toconvert HMW DNA into a Whole Methylome Library competent foramplification by PCR, transcription, or strand displacement, forexample.

Reagent Enz-O-Mix 10 may comprise one of Stem-Loop Oligo AttachmentMaster Mixes shown in FIG. 18, and it is also supplemented with a DNAfragmentation endonuclease such as restriction enzyme, DNase I,benzonase, methylation-specific nuclease McrBC, apoptotic endonuclease,and a mixture of several (about 5-12) methylation-sensitive restrictionenzymes, such as Aci I, Acc II, Asp LE I, Ava I, Bce AI, Bsa HI, Bsh1236 I, Bsi E1, Bsi SI, Bst FN I, Bst HH I, Bst UI, Cfo I, Hap II, HgaI, Hha I, HinP1 I, Hin 6I, Hpa II, Hpy 99I, Hpy CH4 IV, Hsp AI, Mvn I,and Ssi I, to produce a substantial fragmentation of all non-methylatedCpG islands (see, for example, U.S. patent application Ser. No.11/071,864, filed Mar. 3, 2005, which is incorporated by referenceherein in its entirety). It could be also supplemented with athermophilic DNA polymerase. The Methylome library prepared by theEnz-O-Mix 10 process can be used for PCR-mediated WMA in a open-tube(two-step process) or a close-tube (one-step process) formats (as shownin FIG. 30).

Reagent Enz-O-Mix 11 may comprise Stem-Loop Oligo Attachment MasterMixes shown in FIG. 18 (in this case a stem-loop oligonucleotide has theT7 promoter sequence in the loop or stem region in addition to othernecessary base/backbone modifications), and it is also supplemented witha DNA fragmentation endonuclease, such as restriction enzyme, DNase I,benzonase, methylation-specific nuclease McrBC, apoptotic endonuclease,etc.;; and a mixture of several (about 5-about 12) methylation-sensitiverestriction enzymes, such as Aci I, Acc II, Asp LE I, Ava I, Bce AI, BsaHI, Bsh 1236 I, Bsi E1, Bsi SI, Bst FN I, Bst HH I, Bst UI, Cfo I, HapII, Hga I, Hha I, HinP1 I, Hin 6I, Hpa II, Hpy 99I, Hpy CH4 IV, Hsp AI,Mvn I, and Ssi I, to produce a substantial fragmentation at allnon-methylated CpG islands (see, for example, U.S. patent applicationSer. No. 11/071,864, filed Mar. 3, 2005, which is incorporated byreference herein in its entirety). Reagent Enz-O-Mix 11 could be alsosupplemented with T7 RNA polymerase and rNTPs. The Enz-O-Mix 11 can beused for a two-step transcription-mediated Whole Methylome Amplification(when the Whole Methylome library is prepared first, and then amplified)or for a one-step ithothermal amplification process when the Methylomelibrary synthesis and its amplification by transcription occurssimultaneously.

Reagent Enz-O-Mix 12 may comprise Stem-Loop Oligo Attachment MasterMixes shown in FIG. 18 (in this case, a stem-loop oligonucleotide has arecognition sequence for a nicking endonuclease in the loop or stemregion in addition to other necessary base/backbone modifications), andit is also supplemented with a DNA fragmentation endonuclease, such asrestriction enzyme, DNase I, benzonase, methylation-specific nucleaseMcrBC, apoptotic endonuclease; and a mixture of several (about 5-12)methylation-sensitive restriction enzymes, such as Aci I, Acc II, Asp LEI, Ava I, Bce AI, Bsa HI, Bsh 1236 I, Bsi E1, Bsi SI, Bst FN I, Bst HHI, Bst UI, Cfo I, Hap II, Hga I, Hha I, HinP1 I, Hin 6I, Hpa II, Hpy99I, Hpy CH4 IV, Hsp AI, Mvn I, and Ssi I, to produce a substantialfragmentation of all non-methylated CpG islands (see, for example, U.S.patent application Ser. No. 11/071,864, filed Mar. 3, 2005, which isincorporated by reference herein in its entirety). Reagent Enz-O-Mix 12could be also supplemented with nicking endonuclease (N.AlwI, N.BbvCIA,N.BbvCIB, Nb.Bpu10I, Nb.BsmI, N.Bst9I, N.BstNBI, etc.), a stranddisplacing DNA polymerase (Klenow exo-, Phi 29, Bst I, etc.), and primerP (FIG. 15). The Enz-O-Mix 12 can be used for a two-step stranddisplacement-mediated Whole Methylome Amplification (when the library isprepared first, and then amplified) or for a one-step ithothermalamplification process when the Methylome library synthesis and itsamplification by a strand displacement synthesis occurs simultaneously(as shown in FIG. 15).

S. Enz-O-Mix Reagents and Procedures for the One-Step Whole GenomeLibrary Synthesis for Cloning

DNA cloning is a powerful tool for whole genome DNA sequencing andanalysis of cDNA libraries, for example. In a standard process, DNA israndomly fragmented, repaired, attached to linkers, and then integratedinto a vector. This embodiment describes the Enz-O-Mix reagents for theone-step preparation of DNA fragments with sticky ends that is easy toclone.

FIG. 19E shows schematically exemplary compositions of two Enz-O-Mixreagents designed to convert fragmented (Enz-O-Mix 13) or HMW DNA(Enz-O-Mix 14) into a Whole Genome Library for cloning.

Reagent Enz-O-Mix 13 may comprise a Stem-Loop Oligo Attachment MasterMix IV shown in FIG. 18, and it is the only component of the Enz-O-Mix13 reagent. The library generated by the Enz-O-Mix 13 process can onlyutilize a fragmented DNA (for example, cell-free DNA from blood and/orurine, or DNA fragmented enzymatically or mechanically, for example) andbe used for cloning.

Reagent Enz-O-Mix 14 may comprise a Stem-Loop Oligo Attachment MasterMix IV shown in FIG. 18 that is supplemented with a DNA fragmentationendonuclease, such as restriction enzyme, DNase I, benzonase,methylation-specific nuclease McrBC, apoptotic endonuclease, etc. Thelibrary generated by the Enz-O-Mix 14 process can utilize HMW DNA fromany type of cells and tissues and be used for cloning.

T. Enz-O-Mix Reagent Kits and their Use in High-Throughput Applicationsand Clinical Diagnostics

In this embodiment of the present invention, as illustrated in FIG. 20,pre-assembled Enz-O-Mix reagents can be stored at −20° C. and used asready-to-use kits.

Exemplary pre-assembled Enz-O-Mix 1 comprises all major components ofthe reaction, such as a stem-loop oligonucleotide; DNA polymerase; a DNAligase; dU-glycosylase (when the hairpin non-replicable region isgenerated by abasic sites); a hairpin-specific endonuclease (when thehairpin has a cleavable site generated during oligonucleotide attachmentprocess); thermophilic DNA polymerase (close-tube WGA librarysynthesis/PCR-mediated amplification reaction); ATP; and dNTPs.

Exemplary pre-assembled Enz-O-Mix 2 comprises a stem-loopoligonucleotide with a promoter sequence in a stem or loop region; DNApolymerase; a DNA ligase; dU-glycosylase (when the non-replicable regionis formed by abasic sites); a site-specific endonuclease (when thestem-loop oligonucleotide has a cleavable site created duringoligonucleotide attachment process); ATP; and dNTPs; In the case ofclose-tube, isothermal library synthesis/transcription-mediatedamplification reaction it also comprises RNA polymerase and rNTPs.

Exemplary pre-assembled Enz-O-Mix 3 may comprise a stem-loopoligonucleotide with a nicking endonuclease sequence in a stem or a loopregion; DNA polymerase; a DNA ligase; dU-glycosylase (when thenon-replicable region is formed by abasic sites); a site-specificendonuclease (when the stem-loop oligonucleotide has a cleavable sitegenerated during oligonucleotide attachment process); a DNAfragmentation endonuclease, such as restriction enzyme(s), DNase I,Benzonase, methylation-specific nuclease McrBC, apoptotic endonucleaseetc.; ATP; and dNTP, In the case of close-tube, isothermal librarysynthesis/strand displacement-mediated amplification reaction it alsocomprises a nicking endonuclease (N.AlwI, N.BbvCIA, N.BbvCIB, Nb.Bpu10I,Nb.BsmI, N.Bst9I, N.BstNBI, etc.); a strand displacing DNA polymerase(Klenow exo-, Phi 29, Bst I, etc.); and a primer.

Exemplary pre-assembled Enz-O-Mix 4 may comprise all major components ofthe reaction, such as a stem-loop oligonucleotide; DNA polymerase; a DNAligase; dU-glycosylase (when the hairpin non-replicable region iscreated by abasic sites); a hairpin-specific endonuclease (when thehairpin has a cleavable site created during oligonucleotide attachmentprocess); a DNA fragmentation endonuclease such as restriction enzyme,DNase I, Benzonase, methylation-specific nuclease McrBC, apoptoticendonuclease; thermophilic DNA polymerase (close-tube WGA librarysynthesis/PCR-mediated amplification reaction); ATP; and dNTPs.

Exemplary pre-assembled Enz-O-Mix 5 may comprise a stem-loopoligonucleotide with a promoter sequence in a stem or loop region; DNApolymerase; DNA ligase; dU-glycosylase (when the non-replicable regionis formed by abasic sites); a site-specific endonuclease (when thestem-loop oligonucleotide has a cleavable site created during theoligonucleotide attachment process); a DNA fragmentation endonuclease,such as restriction enzyme, DNase I, benzonase, methylation-specificnuclease McrBC, apoptotic endonuclease, etc.; ATP; and dNTPs. In thecase of close-tube, isothermal library synthesis/transcription-mediatedamplification reaction it also comprises RNA polymerase and rNTPs.

Exemplary pre-assembled Enz-O-Mix 6 comprises a stem-loopoligonucleotide with a nicking endonuclease sequence in a stem or a loopregion, DNA polymerase, a DNA ligase, dU-glycosylase (when thenon-replicable region is formed by abasic sites), a site-specificendonuclease (when the stem-loop oligonucleotide has a cleavable sitecreated during oligonucleotide attachment process), a DNA fragmentationendonuclease such as restriction enzyme(s), DNase I, Benzonase,methylation-specific nuclease McrBC, apoptotic endonuclease; ATP; anddNTPs. In the case of close-tube, isothermal library synthesis/stranddisplacement-mediated amplification reaction it also comprises a nickingendonuclease (N.AlwI, N.BbvCIA, N.BbvCIB, Nb.Bpu10I, Nb.BsmI, N.Bst9I,N.BstNBI, etc.); a strand displacing DNA polymerase (Klenow exo-, Phi29, Bst I, etc.); and a primer.

Exemplary pre-assembled Enz-O-Mix 7 may comprise all major components ofthe reaction, such as a stem-loop oligonucleotide; DNA polymerase; T4DNA ligase; dU-glycosylase (when the hairpin non-replicable region iscreated by abasic sites); a hairpin-specific endonuclease (when thehairpin has a cleavable site created during oligonucleotide attachmentprocess); a mixture of several (about 5-about 12) methylation-sensitiverestriction enzymes, such as Aci I, Acc II, Asp LE I, Ava I, Bce AI, BsaHI, Bsh 1236 I, Bsi E1, Bsi SI, Bst FN I, Bst HH I, Bst UI, Cfo I, HapII, Hga I, Hha I, HinP1 I, Hin 6I, Hpa II, Hpy 99I, Hpy CH4 IV, Hsp AI,Mvn I, and Ssi I, to produce a substantial fragmentation at allnon-methylated CpG islands (see, for example, U.S. patent applicationSer. No. 11/071,864, filed Mar. 3, 2005, which is incorporated byreference herein in its entirety); thermophilic DNA polymerase(close-tube WMA library synthesis/PCR-mediated amplification reaction);ATP; and dNTPs.

Exemplary pre-assembled Enz-O-Mix 8 may comprise a stem-loopoligonucleotide with a promoter sequence in a stem or loop region; DNApolymerase; a DNA ligase; dU-glycosylase (when the non-replicable regionis formed by abasic sites); a site-specific endonuclease (when thestem-loop oligonucleotide has a cleavable site created during theoligonucleotide attachment process); a mixture of several (about 5-about12) methylation-sensitive restriction enzymes, such as Aci I, Acc II,Asp LE I, Ava I, Bce AI, Bsa HI, Bsh 1236 I, Bsi E1, Bsi SI, Bst FN I,Bst HH I, Bst UI, Cfo I, Hap II, Hga I, Hha I, HinP1 I, Hin 6I, Hpa II,Hpy 99I, Hpy CH4 IV, Hsp AI, Mvn I, and Ssi I, to produce a substantialfragmentation at all non-methylated CpG islands (see, for example, U.S.patent application Ser. No. 11/071,864, filed Mar. 3, 2005, which isincorporated by reference herein in its entirety); ATP; and dNTPs. Inthe case of close-tube, isothermal librarysynthesis/transcription-mediated amplification reaction it alsocomprises RNA polymerase and rNTPs.

Exemplary pre-assembled Enz-O-Mix 9 may comprise a stem-loopoligonucleotide with a nicking endonuclease sequence in a stem or a loopregion; DNA polymerase; a DNA ligase; dU-glycosylase (when thenon-replicable region is formed by abasic sites); a site-specificendonuclease (when the stem-loop oligonucleotide has a cleavable sitecreated during oligonucleotide attachment process); a DNA fragmentationendonuclease, such as restriction enzyme(s), DNase I, Benzonase,methylation-specific nuclease McrBC, apoptotic endonuclease, etc.; amixture of several (about 5-about 12) methylation-sensitive restrictionenzymes, such as Aci I, Acc II, Asp LE I, Ava I, Bce AI, Bsa HI, Bsh1236 I, Bsi E1, Bsi SI, Bst FN I, Bst HH I, Bst UI, Cfo I, Hap II, HgaI, Hha I, HinP1 I, Hin 6I, Hpa II, Hpy 99I, Hpy CH4 IV, Hsp AI, Mvn I,and Ssi I, to produce a substantial fragmentation at all non-methylatedCpG islands (see, for example, U.S. patent application Ser. No.11/071,864, filed Mar. 3, 2005, which is incorporated by referenceherein in its entirety); ATP; and dNTPs. In the case of close-tube,isothermal library synthesis/strand displacement-mediated amplificationreaction it also comprises a nicking endonuclease (N.AlwI, N.BbvCIA,N.BbvCIB, Nb.Bpu10I, Nb.BsmI, N.Bst9I, N.BstNBI, etc.); a stranddisplacing DNA polymerase (Klenow exo-, Phi 29, Bst I, etc.); and aprimer.

Exemplary pre-assembled Enz-O-Mix 10 may comprise all major componentsof the reaction, such as a stem-loop oligonucleotide; DNA polymerase; aDNA ligase; dU-glycosylase (when the hairpin non-replicable region iscreated by abasic sites); a hairpin-specific endonuclease (when thehairpin has a cleavable site created during oligonucleotide attachmentprocess); a DNA fragmentation endonuclease, such as restriction enzyme,DNase I, Benzonase, methylation-specific nuclease McrBC, apoptoticendonuclease, etc.; a mixture of several (about 5-about 12)methylation-sensitive restriction enzymes, such as Aci I, Acc II, Asp LEI, Ava I, Bce AI, Bsa HI, Bsh 1236 I, Bsi E1, Bsi SI, Bst FN I, Bst HHI, Bst UI, Cfo I, Hap II, Hga I, Hha I, HinP1 I, Hin 6I, Hpa II, Hpy99I, Hpy CH4 IV, Hsp AI, Mvn I, and Ssi I, to produce a substantialfragmentation at all non-methylated CpG islands (see, for example, U.S.patent application Ser. No. 11/071,864, filed Mar. 3, 2005, which isincorporated by reference herein in its entirety); thermophilic DNApolymerase (close-tube WGA library synthesis/PCR-mediated amplificationreaction); ATP; and dNTPs.

Exemplary pre-assembled Enz-O-Mix 11 may comprise a stem-loopoligonucleotide with a promoter sequence in a stem or loop region; DNApolymerase; a DNA ligase; dU-glycosylase (when the non-replicable regionis formed by abasic sites); a site-pecific endonuclease (when thestem-loop oligonucleotide has a cleavable site created duringoligonucleotide attachment process); a DNA fragmentation endonuclease,such as restriction enzyme, DNase I, benzonase, methylation-specificnuclease McrBC, apoptotic endonuclease; a mixture of several (about5-12) methylation-sensitive restriction enzymes, such as Aci I, Acc II,Asp LE I, Ava I, Bce AI, Bsa HI, Bsh 1236 I, Bsi E1, Bsi SI, Bst FN I,Bst HH I, Bst UI, Cfo I, Hap II, Hga I, Hha I, HinP1 I, Hin 6I, Hpa II,Hpy 99I, Hpy CH4 IV, Hsp AI, Mvn I, and Ssi I, to produce a substantialfragmentation at all non-methylated CpG islands (see, for example, U.S.patent application Ser. No. 11/071,864, filed Mar. 3, 2005, which isincorporated by reference herein in its entirety); ATP; and dNTPs. Inthe case of close-tube, isothermal librarysynthesis/transcription-mediated amplification reaction it alsocomprises RNA polymerase and rNTPs.

Exemplary pre-assembled Enz-O-Mix 12 may comprise a stem-loopoligonucleotide with a nicking endonuclease sequence in a stem or a loopregion; DNA polymerase; a DNA ligase; dU-glycosylase (when thenon-replicable region is formed by abasic sites); a site-specificendonuclease (when the stem-loop oligonucleotide has a cleavable sitecreated during oligonucleotide attachment process); a DNA fragmentationendonuclease, such as restriction enzyme(s), DNase I, benzonase,methylation-specific nuclease McrBC, apoptotic endonuclease; a mixtureof several (about 5-about 12) methylation-sensitive restriction enzymes,such as Aci I, Acc II, Asp LE I, Ava I, Bce AI, Bsa HI, Bsh 1236 I, BsiE1, Bsi SI, Bst FN I, Bst HH I, Bst UI, Cfo I, Hap II, Hga I, Hha I,HinP1 I, Hin 6I, Hpa II, Hpy 99I, Hpy CH4 IV, Hsp AI, Mvn I, and Ssi I,to produce a substantial fragmentation at all non-methylated CpG islands(see, for example, U.S. patent application Ser. No. 11/071,864, filedMar. 3, 2005, which is incorporated by reference herein in itsentirety); ATP; and dNTPs. In the case of close-tube, isothermal librarysynthesis/strand displacement-mediated amplification reaction it alsocomprises a nicking endonuclease (N.AlwI, N.BbvCIA, N.BbvCIB, Nb.Bpu10I,Nb.BsmI, N.Bst9I, N.BstNBI, etc.) a strand displacing DNA polymerase(Klenow exo-, Phi 29, Bst I, etc.); and a primer.

Kits comprising the exemplary Enz-O-Mixes described above may besupplied with the Enz-O-Mix Universal Buffer. When used inhigh-throughput applications and clinical diagnostics, the Enz-O-Mixreagent is first diluted by the Enz-O-Mix Universal Buffer, aliquotedinto microplate wells, pre-incubated at the reaction temperature (forexample, about 37° C.) for a few minutes, supplemented with DNA, andincubated at the reaction temperature for about 1-about 16 h.

FIG. 20 demonstrates an exemplary application of the Enz-O-Mix kits forhigh-throughput library preparation/amplification in the 96- or 384-wellformat.

U. Enz-O-Mix Reaction Optimization Procedures

In this embodiment, as shown in FIG. 21, there are exemplary proceduresfor identifying the most optimal parameters for any Enz-O-Mix processusing real-time PCR to monitor amplification of the generated library,or a direct fluorescent detection of the amplified product in the caseof combined isothermal library synthesis-amplification process.

FIG. 21A illustrates the procedure for optimization of the Enz-O-MixUniversal Buffer. Three specific but exemplary buffers are utilized,including Universal Buffers 1, 2 and 3. Buffer 2 results in the earlyWGA amplification curve, suggesting that the multi-enzymatic reactionwas more efficient in this buffer, and the Universal Buffer 2 isparticularly suitable for the Enz-O-Mix reaction.

FIG. 21B illustrates the procedure for concentration optimization of theEnz-O-Mix incubation temperature. Three temperatures are employed;specifically, T₁, T₂ and T₃. Temperature T₃ results in the early WGAamplification curve, suggesting that the multi-enzymatic reaction wasmore efficient at this temperature and that the parameter T₃ should beselected as a more suitable condition for the Enz-O-Mix reaction.

FIG. 21C illustrates the procedure for optimization of the Enz-O-Mixcomponent N. Three concentrations of the component N; specifically,C^(N) ₁, C^(N) ₂ and C^(N) ₃, were utilized. Concentration C^(N) ₁results in the early WGA amplification curve, suggesting that themulti-enzymatic reaction was more efficient at this component Nconcentration, and that the parameter C^(N) ₁ is a particularly suitablecondition for the Enz-O-Mix reaction.

V. High Potential for the Enz-O-Mix Approach in Future Biotechnology andDiagnostic Medicine

The applications of the Enz-O-Mix method are numerous. The Enz-O-Mixmethod is easy to automate and use in clinical diagnostic andpoint-of-care applications. Different enzyme combinations could lead tomany novel kits and assays, and applications in such areas asbiotechnology, the pharmaceutical industry, molecular diagnostics,forensics, pathology, bio-defense, and bio-computing are contemplatedThe Enz-O-Mix approach can be viewed as a simple and cost-effectivealternative to the “lab-on-a chip” micro-fluidic approach that iscurrently attempting to solve the same problem (multi-step DNAprocessing) by reduction of reaction volumes and integration of multiplereactions into a small format.

Exemplary applications for the invention include but are not limited tothe following:

a) A one-step, closed tube preparation and amplification of genomiclibraries (WGA) from highly degraded serum, plasma, and/or urine (suchas the supernatant fraction) DNA. DNA amplification and re-amplificationcan be used as an in vitro “immortalization” process to maintain andgenerate necessary quantities of valuable but limited DNA samples forgene association studies, mutation and microsatellite instabilitydetection in cancer diagnostics, etc.

b) A one-step, closed tube preparation and amplification of genomiclibraries (WGA) from formalin fixed, paraffin embedded tissues forcancer diagnostics and research applications.

c) A one-step, closed tube preparation and amplification of Methylomelibraries;

d) A one-step preparation and simultaneous immobilization of preparedDNA libraries on a solid support.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples that follow representtechniques discovered by the inventors to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments that are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 One Step Whole Genome Library Amplification Using HairpinAdaptor with EcoNI Restriction Site Generated after Ligation andStrand-Displacement Extension

In this example, a single step WGA process is described wherein apartial hairpin oligonucleotide adaptor containing EcoNI recognitionhalf sites in its stem is extended by a DNA polymerase to form a bluntend hairpin molecule and is ligated via its free 3′ end to the 5′phosphate of blunt-ended DNA restriction fragments by T4 DNA ligase. Dueto the strand-displacement activity of the DNA polymerase, the free 3′end of the restriction fragments are extended using as template theligated 3′ end of the adaptor, thus opening up the hairpin structure andgenerating functional EcoNI cleavage site (FIGS. 12 and 13A). Followingcleavage with EcoNI present in the same reaction mix, the DNA librarymolecules have their terminal inverted repeats excised and becomeamplifiable by PCR using a primer complementary to the 5′ stem portionof the adaptor sequence. The whole process takes place in a single tubeand in one step in just about 1 hour, in specific embodiments.

Human genomic DNA isolated from the peripheral blood of a healthy donorby standard procedures was digested with 10 units of AluI restrictionenzyme (NEB) for 1 hour following the manufacturer's protocol.

For library preparation, 5 nanograms of AluI-digested DNA were incubatedin a reaction mixture comprising 1× NEBuffer 4 (20 mM Tris-acetate, 10mM Mg acetate, 50 mM potassium acetate, 1 mM dithiothreitol, pH 7.9), 1μM EcoNI Ext Adaptor oligonucleotide (Table I, SEQ ID NO:1), 1 mM ATP,120 μM dNTPs, 2 Units of Klenow fragment of DNA polymerase I, 400 unitsof T4 DNA ligase (New England Biolabs, NEB; Beverly, Mass.), and 10units of EcoNI restriction enzyme (NEB) in a final volume of 15 μl for 1hour at 37° C. Enzymes were inactivated for 10 min at 75° C. Controlreaction that contained no EcoNI restriction enzyme was also run inparallel.

The resulting library was amplified by real-time PCR in a reactionmixture containing: 1× Titanium Taq reaction buffer (Clontech; MountainView, Calif.); 200 μM each dNTP; 1:10,000 dilutions of fluorescein andSybrGreen I (Molecular Probes; Carlsbad, Calif.); 1 μM of K_(U) primer(Table I, SEQ ID NO:2); and 5 units of Titanium Taq polymerase(Clontech; Mountain View, Calif.) in a reaction volume of 75 μA PCRamplification was carried out for 15 cycles at 95° C. for 15 sec and 65°C. for 2 min on i-Cycler real-time PCR instrument (Bio-Rad; Hercules,Calif.). FIG. 22 shows the amplification curves of the librariesprepared in the presence or in the absence of EcoNI. Cleavage of theterminal inverted repeat by EcoNI improved the efficiency ofamplification by 7 cycles, i.e. over two orders of magnitude.

Preliminary representation analysis of the library prepared in thepresence of EcoNI was conducted using three exemplary human genomic STSmarkers (Table II). The material amplified by PCR with the universalK_(U) primer was purified with Qiaquick filters (Qiagen; Valencia,Calif.), and 20 ng aliquots were analyzed in real-time PCR. Reactionswere carried out for 45 cycles at 94° C. for 15 sec and 68° C. for 1 minon i-Cycler (Bio-Rad; Hercules, Calif.). The PCR reaction mixturecomprised the following: 1× Titanium Taq reaction buffer (Clontech;Mountain View, Calif.); 200 μM each dNTP; 1:10,000 dilutions offluorescein and SybrGreen I (Molecular Probes); 0.2 μM each forward andreverse STS primer (Table II); 5 units of Titanium Taq polymerase(Clontech; Mountain View, Calif.); and 20 ng library DNA. A controlreaction comprising 1 ng of human genomic DNA randomly fragmented to anaverage size of 1.5 Kb using Hydro Shear device (Gene Machines; PaloAlto, Calif.) was performed. FIGS. 24A-24C show unbiased representationof the analyzed human STS sequences in the EcoNI-treated library.

Example 2 One Step Whole Genome Amplification Using Hairpin Adaptor withNon-Replicable 18-Atom Hexa-Ethyleneglycol Spacer

In this example, a single step WGA process is described wherein ahairpin oligonucleotide adaptor containing non-replicable 18-atomhexa-ethyleneglycol (HEG) spacer in its loop is ligated via its free 3′end to the 5′ phosphate of DNA restriction fragments in the presence ofT4 DNA ligase and a DNA polymerase. Due to the strand-displacementactivity of the polymerase, the free 3′ ends of the restrictionfragments are extended until the replication stop is reached (see FIGS.8 and 10A). This process results in truncated 3′ termini of theresulting WGA library such that they do not contain a terminal invertedrepeat. The library molecules are then amplified by PCR using a primercomplementary to the universal sequence of the extended 3′ termini. Thewhole process takes place in a single tube in one step and is completedin just about 1 hour, in specific embodiments.

In control experiments, hairpin adaptor molecules that either do notcomprise a replication stop or modified bases or that compriseribo-nucleosides in their loop region are compared for their ability toparticipate in WGA along with the hexa-ethyleneglycol adaptor describedabove.

Human genomic DNA isolated from the peripheral blood of a healthy donorby standard procedures was digested with 10 units of AluI restrictionenzyme (NEB) for 1 hour following the manufacturer's protocol.

Five nanograms of AluI digested DNA were incubated in a reaction mixturecomprising 1× NEBuffer 4; 1 μM HEG Adaptor oligonucleotide (Table I, SEQID NO:3) or control hairpin adaptors (see below); 1 mM ATP; 120 μMdNTPs; 2 Units of Klenow fragment of DNA polymerase I; and 400 units ofT4 DNA ligase (NEB) in a final volume of 15 μl for 1 hour at 37° C.Enzymes were inactivated for 10 min at 75° C.

To test the effect of the hexa-ethyleneglycol replication stop, controlreactions containing partial hairpin oligonucleotides extended by Klenowto form blunt end hairpin adaptors that either do not containreplication stop (Table I, Hairpin Adaptor, SEQ ID NO:5), or containingribonucleosides in their loop region (Table I, Hairpin Ribo Adaptor, SEQID NO:6) were substituted for the HEG Adaptor (Table I, SEQ ID NO:3) andrun in parallel as described above.

The resulting libraries were amplified by real-time PCR in a reactionmixture comprising the following: 1× Titanium Taq reaction buffer(Clontech; Mountain View, Calif.); 200 μM each dNTP; 1:10,000 dilutionsof fluorescein and SybrGreen I (Molecular Probes; Carlsbad, Calif.); 1μM of universal primer (Y_(U) primer, Table I SEQ ID NO:4 in the case ofthe HEG adaptor, or K_(U) primer, Table I, SEQ ID NO:2, in the case ofcontrol hairpin adaptors); and 5 units of Titanium Taq polymerase(Clontech; Mountain View, Calif.) in a reaction volume of 75 μl. PCRamplification was carried out at 95° C. for 15 sec and 65° C. for 2 minon i-Cycler real-time PCR instrument (Bio-Rad; Hercules, Calif.). Asshown on FIG. 23, amplification of the library prepared by ligation ofhexa-ethyleneglycol adaptor was 9 cycles more efficient than a librarymade with adaptor not containing a replication stop in its loopstructure. The results shown on FIG. 23 also indicate that an array of 5consecutive ribonucleosides in the adaptor's loop is not a preferablereplication stop for Klenow fragment of DNA polymerase I, sinceamplification of a library prepared by ligation of such an adaptoramplified only 2 cycles earlier than a library made with adaptorcontaining only deoxyribonucleosides.

Preliminary representation analysis of the library prepared by ligationof HEG adaptor was done using three exemplary human genomic STS markers(Table II). The material amplified by PCR with the universal Y_(U)primer was purified with Qiaquick filters (Qiagen; Valencia, Calif.),and 20 ng aliquots were analyzed in real-time PCR as described inExample 1. FIGS. 24A-24C show unbiased representation of the analyzedhuman STS sequences in this exemplary library.

Example 3 One Step Whole Genome Library Preparation Using DegradableHairpin Adaptor Comprising Deoxy-Uridine

In this example, a single step WGA process is described wherein ahairpin oligonucleotide adaptor comprising deoxy-uridine in both its 5′stem region and in its loop (FIG. 10B) is ligated via its free 3′ end tothe 5′ phosphates of DNA restriction fragments in the presence of 3enzymatic activities: T4 DNA ligase, DNA polymerase, and Uracil-DNAglycosylase (which is also referred to as UDG or dU glycosylase). UDGcatalyzes the release of free uracil and generates abasic sites in theadaptor's loop region and the 5′ half of the hairpin. Thestrand-displacement activity of the DNA polymerase extends the free 3′end of the restriction fragments until an abasic site is reached,serving as a replication stop (FIG. 10B). This process results intruncated 3′ ends of the resulting WGA library fragments such that theydo not have terminal inverted repeats. Prior to amplification, samplesare heated in order to break the phosphodiester bonds of the generatedabasic sites, and the adaptor molecules are degraded into 2 smallfragments and one intact 18 base oligonucleotide complementary to theuniversal sequence of the extended 3′ termini (FIG. 10B). The released18 base oligonucleotide then serves as a primer in subsequent PCRamplification, or alternatively it can be exogenously supplied. Theentire process takes place in a single tube in one step and is completedin just 1 hour.

Human genomic DNA isolated from the peripheral blood of a healthy donorby standard procedures was digested with 10 units of AluI restrictionenzyme (New England Biolabs; Beverly, Mass.) for 1 hour following themanufacturer's protocol.

For library preparation, five nanograms of AluI-digested genomic DNAwere incubated in an exemplary reaction mixture comprising 1× NEBuffer4; 2 μM dU Hairpin Adaptor oligonucleotide (Table I, SEQ ID NO:7); 1 mMATP; 120 μM dNTPs; 2 Units of Klenow fragment of DNA polymerase I; 400units of T4 DNA ligase; and 2 Units of UDG (New England Biolabs;Beverly, Mass.) in a final volume of 15 μl for 1 hour at 37° C. Enzymeswere inactivated at 75° C. for 10 min. A control reaction containing noUDG was run in parallel.

The resulting library was amplified by real-time PCR in a reactionmixture

comprising the following: 1× Titanium Taq reaction buffer (Clontech,Mountain View, Calif.); 200 μM each dNTP; 1:10,000 dilutions offluorescein and SybrGreen I (Molecular Probes; Carlsbad, Calif.); 1 μMof universal M_(U) primer (Table I, SEQ ID NO:8); and 5 units ofTitanium Taq polymerase (Clontech; in a reaction volume of 75 μl). PCRamplifications were carried out at 95° C. for 15 sec and 65° C. for 2min on i-Cycler real-time PCR instrument (Bio-Rad; Hercules, Calif.). Ina separate amplification reaction, no external universal M_(U) primerwas supplied to test if endogenously-generated primer is sufficient forlibrary amplification. FIG. 25 shows that virtually identicalamplification curves were generated whether or not the universal primerwas added exogenously or was generated as a result of the UDG activityfollowed by heat-induced degradation of the adaptor. FIG. 25 also showsthat if UDG was not present during library preparation, this caused an 8cycle-delay in amplification. This latter result once again indicatesthe preference of removal of the terminal inverted repeat of a hairpinadaptor for efficient library priming and amplification.

Example 4 One Step Genomic DNA Restriction Digestion and Whole GenomeLibrary Preparation Using Degradable Hairpin Adaptor ComprisingDeoxy-Uridine

In this example, a single step WGA process is described wherein ahairpin oligonucleotide adaptor comprising deoxy-uridine described inExample 3 is ligated via its free 3′ end to the 5′ phosphates of DNArestriction fragments generated from intact genomic DNA in a singleexemplary reaction mix comprising 4 exemplary enzymatic activities: AluIrestriction endonuclease, T4 DNA ligase, DNA polymerase, and Uracil-DNAglycosylase (UDG) (FIG. 19A, Enz-O-Mix 3). UDG catalyses the release offree uracil and generates abasic sites in the adaptor's loop region andthe 5′ half of the hairpin. AluI digests the target DNA into restrictionfragments. T4 DNA ligase generates a phosphodiester bond between the 3′ends of the adaptor molecules and the 5′ phosphates of the restrictionfragments. Finally, the strand-displacement activity of the DNApolymerase extends the free 3′ end of the restriction fragments using astemplate the ligated 3′ end of the hairpin stem until an abasic site isreached that serves as a replication stop (FIG. 10B). Prior toamplification, samples are heated to degrade the abasic sites of theadaptor, and the resulting library is amplified using the universalprimer M_(U). The entire process takes place in a single tube in onestep and is completed in just 1 hour.

For library preparation, 20, 10, 3, 1, 0.3, 0.1, 0.03, 0.01, or 0nanograms of human genomic DNA isolated from the peripheral blood of ahealthy donor by standard procedures were incubated in an exemplaryreaction mix comprising: 1× NEBuffer4; 2 μM dU Hairpin Adaptoroligonucleotide (Table I, SEQ ID NO:7); 1 mM ATP; 120 μM dNTPs; 2 Unitsof Klenow fragment of DNA polymerase I; 400 units of T4 DNA ligase; 2Units of UDG; and 10 units of AluI restriction endonuclease (New EnglandBiolabs; Beverly, Mass.) in a final volume of 15 μl for 1 hour at 37° C.Enzymes were inactivated for 10 min at 75° C.

The resulting libraries were amplified by real-time PCR in the exemplaryreaction mixture comprising the following: 1× Titanium Taq reactionbuffer (Clontech; Mountain View, Calif.); 200 μM each dNTP; 1:10,000dilutions of fluorescein and SybrGreen I (Molecular Probes; Carlsbad,Calif.); 1 μM of universal M_(U) primer (Table I, SEQ ID NO:8); and 5units of Titanium Taq polymerase (Clontech; Mountain View, Calif.) in areaction volume of 75 μl. PCR amplifications were carried out at 95° C.for 15 sec and 65° C. for 2 min on i-Cycler real-time PCR instrument(Bio-Rad; Hercules, Calif.). FIG. 26 shows the amplification curves ofthe resulting libraries. As shown, a series of titration curves wasgenerated in a concentration-dependent manner. The smallest amount ofDNA corresponding to less than two genome equivalents was stilldiscernible from the control reaction with no DNA, indicating that thedescribed single closed tube process is an efficient and sensitive wayof whole genome amplification.

Example 5 Simplified Protocol Combining Preparation of MethylomeLibraries from Cell-Free Urine DNA and Cleavage withMethylation-Sensitive Restriction Enzymes in One Step

In this example, the preparation of methylome libraries from cell-freeurine DNA by ligation of hairpin oligonucleotide adaptor comprisingdeoxy-uridine as described in Example 3 is combined with thesimultaneous cleavage with a mix of methylation-sensitive restrictionenzymes in a single step.

Cell-free DNA was isolated from urine of healthy donors collected in 50ml Falcon tubes and stabilized for storage by adding 0.1 volume of 0.5 MEDTA. Urine samples were centrifuged at 1,800×g for 10 min at ambienttemperature to sediment cells, and supernatant was transferred carefullyto a fresh tube. Equal volume of 6 M solution of guanidine thiocyanatewas added to each sample followed by 1/6 vol of Wizard Miniprep resin(Promega catalog #A7141; Madison, Wis.). DNA was bound to the resin byrotation for 1 hour at ambient temperature. The resin was thensedimented by brief centrifugation at 500×g and loaded on Wizardminicolumns (Promega catalog #A7211; Madison, Wis.) using syringe barrelextensions after carefully decanting out the supernatant. Resin waswashed with 5 ml of wash buffer (Promega catalog #A8102; Madison, Wis.)using Qiagen QJAvac-24 vacuum manifold. Minicolumns were thencentrifuged for 2 min at 10,000×g to remove residual wash buffer andbound DNA was eluted with 50 μl of DNAse-free water at 10,000×g for 1min. Eluted DNA was buffered by adding 0.1 vol of 10×TE-L buffer andquantified on a fluorescent spectrophotometer using Pico Green(Molecular Probes; Carlsbad, Calif.) and 1 phage D.

Aliquots of 200 ng of cell-free DNA were artificially methylated byincubation in 20 μl of NEBuffer 2 (New England Biolabs; Beverly, Mass.)with 4 units of M.SssI CpG methylase (New England Biolabs; Beverly,Mass.) in the presence of 160 μM SAM for 1 hour at 37° C.

Twenty five nanograms of methylated or non-methylated DNA were incubatedin 1× NEBuffer 4 (New England Biolabs; Beverly, Mass.) comprising 0.35units of T4 DNA polymerase (New England Biolabs; Beverly, Mass.); 1.5 μMof dU-Hairpin Adaptor (Table I, SEQ ID NO:7); 0.5 units of UDG (NewEngland Biolabs; Beverly, Mass.); 400 units of T4 DNA ligase (NewEngland Biolabs; Beverly, Mass.); 30 μM dNTPs, 0.75 mM ATP; 75 μg/mlBSA; 16.7 units of AciI; 16.7 units of HhaI; 8.3 units each of BstUI,HpaII, and Hinp1I (New England Biolabs; Beverly, Mass.) in a finalvolume of 20 μl for 1 hour at 37° C. A second aliquot of 25 ng ofmethylated or non-methylated DNA was incubated in parallel with all theingredients described above but without the restriction enzymes (“uncut”control).

One half of each sample (12.5 ng) was then amplified by quantitative PCRin an exemplary reaction mix comprising 1× Titanium Taq reaction buffer(Clontech; Mountain View, Calif.); 200 μM of each dNTP; fluoresceincalibration dye (1:100,000) and SYBR Green I (1:100,000); 1 μM universalprimer M_(U)-1 (Table I, SEQ ID NO:8); 4% DMSO; 200 μM 7-deaza-dGTP(Sigma); and 5 units of Titanium Taq polymerase (Clontech) in a finalvolume of 75 μl. Samples were pre-heated at 72° C. for 15 min followedby 95° C. for 5 min and 12 cycles at 94° C. for 15 sec and 65° C. for 2min on an I-Cycler real-time PCR instrument (Bio-Rad; Hercules, Calif.).Amplified libraries were purified using MultiScreen PCR cleanup system(Millipore) and quantified by optical density reading.

Methylation analysis of promoter sites was performed by real-time PCRusing aliquots of 160 ng of each digested or non-digested amplifiedlibrary DNA incubated in exemplary reaction mixtures comprising thefollowing: 1× Titanium Taq reaction buffer (Clontech; Mountain View,Calif.); 200 μM of each dNTP; 4% DMSO; 0.5 M betaine; FCD (1:100,000)and SYBR Green I (1:100,000); 200 nM each forward and reverse primer(Table II, SEQ ID NO:11 and SEQ ID NO:12 for GSTP-1 promoter, SEQ IDNO:13 and SEQ ID NO:14 for MDR-1 promoter, SEQ ID NO:9 and SEQ ID NO:10for EDNRB promoter, and SEQ ID NO:15 and SEQ ID NO:16 for PTGS-2promoter); and approximately 1.5 units of Titanium Taq polymerase(Clontech; Mountain View, Calif.) in a final volume of 15 μl at 95° C.for 3 min followed by 50 cycles at 94° C. for 15 sec and 68° C. for 1min.

FIG. 27 shows PCR amplification curves of specific promoter sites inamplified libraries prepared from methylated (M) or non-methylated (N)urine DNA in the presence (+) or in the absence (−) ofmethylation-sensitive restriction enzymes. As expected, promoter sitesfrom non-methylated cleaved DNA (N+) amplified with significant (atleast 10 cycles) delay as compared to uncut DNA (N−, M−) for all fourpromoter sites tested. On the other hand, methylated DNA was completelyrefractory to cleavage (M+). These results demonstrate that the methoddisclosed in the present invention can be applied as a simple one-stepnon-invasive high-throughput diagnostic procedure for detection ofaberrant methylation in cancer.

TABLE I OLIGONUCLEOTIDE SEQUENCES No Code Sequence 5′-3′  1.EcoNI Ext Adaptor CCAAACACACCCAACACA CCT AAAAA AGG TGT  (SEQ ID NO: 1)* 2. K_(U) TGTGTTGGGTGTGTTTGG  (SEQ ID NO: 2)  3. HEG AdaptorAAGAGAGAGGGAAGGAAGAA/HEG/AACTTCCTTCCCTCTC TCTT (SEQ ID NO: 3)**  4.Y_(U) CTTCCTTCCCTCTCTCTT (SEQ ID NO: 4)  5. Hairpin AdaptorCCAAAC ACACCCAACACAAAAAGTGTTG  (SEQ ID NO: 5)  6. Hairpin Ribo AdaptorCCAAAC ACACCCAACACrArArArArAGTGTTG  (SEQ ID NO: 6)***  7.dU Hairpin Adaptor TGTGTTGGGdUGdUGTGTGGdUdUdUdUdUdUCCACACACACCCAACACA (SEQ ID NO: 7)****  8. M_(U) CCACACACACCCAACACA (SEQ ID NO: 8) 9. EDNRB GGAGGAGTCTTTCGAGTTCAA  (SEQ ID NO: 9) ForwardCGGGAGGAATACAGACACGTCTT  (SEQ ID NO: 10) Reverse 10. GSTP-1GGAAAGAGGGAAAGGCTTC  (SEQ ID NO: 11) Forward CCCCAGTGCTGAGTCACGG (SEQ ID NO: 12) Reverse 11. MDR-1 GGGTGGGAGGAAGCATCGTC (SEQ ID NO: 13) Forward GGTCTCCAGCATCTCCACGAA  (SEQ ID NO: 14) Reverse12. PTGS-2 AGAACTGGCTCTCGGAAGCG  (SEQ ID NO: 15) ForwardGGGAGCAGAGGGGGTAGTC  (SEQ ID NO: 16) Reverse 13. dUT7 Hairpin AdaptorTGTGTTGGGdUGdUGTGTGGdUdUdUdUdUdUATTTAATACGACTCACTATAGGGAGACCACACACACCCAACACA  (SEQ ID NO: 17)**** 14.dU_N.BbvC IB_Nick TGTGTTGGGdUGdUGTGTGGdUdUdUdUdUdUATTTAATACGHairpin Adaptor AC CCTCAGC ACCACACACACCCAACACA  (SEQ ID NO:18)***** 15.dU_N.BbvC IB_Nick ATTTAATACGAC CCTCAGC ACCAC  Primer(SEQ ID NO: 19)***** *Underlined sequences indicate EcoNI palindrome**HEG = hexa-ethyleneglycol ***rA = Adenosine ****dU = deoxy-Uridine*****Underlined sequence indicates N.BbvC IB nicking endonucleaserecognition site

TABLE II HUMAN STS MARKERS USED FOR REPRESENTATION ANALYSIS BYQUANTITATIVE REAL-TIME PCR STS # UniSTS Database Name* 4 SHGC-149956 8csnpmnat1-pcr1-1 35 SHGC-146602 *Unique names of STS marker sequencesfrom the National Center for Biotechnology Information UniSTS database.Sequences of the STS regions as well as the forward and backward primersused in quantitative real-time PCR can be found in the UniSTS databaseat the National Center for Biotechnology Information website.

Example 6 Enz-O-Mix DNA Library Preparation and SimultaneousAmplification by Transcription

This example demonstrates an exemplary preparation of a library fromlambda phage BstEII restriction digest by ligation of hairpinoligonucleotide adaptor comprising deoxy-uridine and T7 promotersequence combined with the simultaneous isothermal amplification by invitro transcription with T7 RNA polymerase (see FIG. 6A and FIG. 19A,Enz-O-Mix 2). The adaptor oligonucleotide used in this example (Table I,SEQ ID NO:9) is based on the adaptor sequence described in Example 3(Table I, SEQ ID NO:8), except that it has the consensus T7 phagepromoter sequence incorporated in the loop region. Four enzymaticactivities are present in the mix: T4 DNA ligase, T4 DNA polymerase,Uracil-DNA glycosylase (UDG), and T7 RNA polymerase. UDG creates abasicsites by destabilizing the 5′ end of the adaptor stem. T4 DNA polymerasegenerates blunt ends at the restriction fragments leaving intact 5′phosphate groups that are necessary for ligation to the free 3′ end ofthe adaptor by the T4 DNA ligase. T4 DNA polymerase then extends the 3′ends of the newly ligated restriction fragments into the adaptor,thereby displacing the 5′ end of the adaptor's stem until an abasic sitecreated by UDG is reached that stops further extension. By doing so, theT4 DNA polymerase generates a fully functional double-stranded T7promoter, and in vitro transcription is initiated by T7 RNA polymerase.

One hundred nanograms of a lambda phage BstEII restriction DNA fragments(NEB Cat. #M0208S; New England Biolabs; Beverly, Mass.) were incubatedin 1× NEBuffer 4 (NEB) with 2 μM adaptor oligonucleotide comprisingdeoxy-uridine and T7 promoter sequence (Table I, SEQ ID NO:9); 1 mM ATP;40 μM each dNTP; 500 μM each rNTP; 200 μg/ml BSA; 5.6 mM DTT; 1 unit ofuracil-DNA glycosylase (New England Biolabs; Beverly, Mass.); 0.18 unitof T4 DNA polymerase (New England Biolabs; Beverly, Mass.); 600 units ofT4 DNA ligase (New England Biolabs; Beverly, Mass.); and 37.5 units ofT7 RNA polymerase (Epicentre; Madison, Wis.) for 2 hours at 37° C. in afinal volume of 15 μl. A negative control containing no T4 DNA ligasewas also run in parallel. Products of the library amplification wereanalyzed by gel electrophoresis on 1.5% agarose gel after staining withSybr Gold (Molecular Probes; Carlsbad, Calif.).

FIG. 28 shows the products of an exemplary in vitro transcriptionamplification. As shown, in the presence of all four enzymaticactivities the lambda restriction fragments are converted into a libraryamplified as RNA products, whereas in the absence of T4 DNA ligase noaccumulation of RNA occurs. This demonstrates that library preparationand isothermal amplification can be combined into a single-step process.

Example 7 Enz-O-Mix DNA Library Preparation and SimultaneousImmobilization on Solid Support

This embodiment is illustrated in FIG. 29 and describes the one-stepEnz-O-Mix attachment process for a stem-loop oligonucleotide with anon-replicable linker that is accompanied by immobilization of thesynthesized library to the surface of a vessel where the reaction occurs(for example, a tube, micro-plate well, glass slide, micro-beads, etc.).The exemplary reaction mix comprises HMW DNA; a stem-loopoligonucleotide with 3′ recessed, 3′ protruding or blunt end (FIG. 4),and a non-replicable linker somewhere in the central part of theoligonucleotide; a DNA fragmentation endonuclease, such as restrictionenzyme, DNase I, benzonase, methylation-specific nuclease McrBC,apoptotic endonuclease, etc.; a 3′ proofreading DNA polymerase (Klenowfragment of the DNA polymerase I, T4 DNA polymerase, etc.); T4 DNAligase; Enz-O-Mix Universal Buffer; ATP; and dNTPs. In specificembodiments, the reaction occurs inside a tube or a micro-plate wellcontaining a hybridization-capture oligonucleotide (HCO) covalentlyattached to the inner surface of the reaction vessel. OligonucleotideHCO has a sequence that is complementary to a portion of the stem-loopoligonucleotide located between the 5′ end and the non-replicablelinker. Four enzymatic reactions and one hybridization reaction aretaking place as follows: (1) DNA fragmentation by a nuclease(s); (2)“polishing” of the DNA ends and the stem-loop oligonucleotidedouble-stranded stem-region; (3) ligation of the oligonucleotide 3′ endto the 5′ phosphate of the DNA, leaving a nick between the 3′ end of DNAand the 5′ end of the oligonucleotide double-stranded stem-region; (4)polymerase extension of the 3′ DNA end that propagates toward the end ofstem-loop oligonucleotide, displaces the 5′ portion of the stem-loopoligonucleotide, stops somewhere within the loop or close to the loopregion, at the replication block, and generates single strandedoverhangs at the 5′ ends of DNA molecules; and (5) immobilization of DNAfragments through a hybridization of the generated 5′ overhangs to thesurface-attached capture oligonucleotide HCO.

Library immobilization can be non-covalent (as shown on FIG. 29A), orcovalent (as shown on FIG. 29B). The first case (non-covalentimmobilization) relies solely on the hybridization between the 5′overhang (produced from the stem-loop oligonucleotide during itsligation to DNA ends and subsequent replication) and the capture oligoHCO. The second case (covalent immobilization) involves hybridization asan initial step, but it also involves additional enzymatic steps, suchas site-specific cleavage within the single stranded 5′ overhang andligation of the 5′ end of DNA to the ligation-capture oligonucleotideconstruct LCO. The purpose of the cleavage reaction is to generate a 5′phosphate group for the ligation reaction. Such cleavage can beachieved, for example, by incubation with the USER enzyme (a mixture ofUracil DNA glycosylase (UDG) and the DNA glycosylase-lyase EndonucleaseVIII; New England Biolabs, Beverly, Mass.). In that case, UDG catalysesthe excision of a uracil base (located somewhere at the 5′ portion ofthe stem-loop oligonucleotide), forming an abasic (apyrimidinic) sitewhile leaving the phosphodiester backbone intact. The lyase activity ofEndonuclease VIII breaks the phosphodiester backbone at the 3′ and 5′sides of the abasic site leaving a 5′ phosphate. A generated phosphategroup at the end of the 5′ overhang is then ligated by a DNA ligase tothe 3′ end of the ligation-capture oligonucleotide construct LCO (seeFIG. 29B).

Library synthesis and immobilization can occur within the specificallydesigned tubes, micro-well plates, on the surface of micro-slides, ormicro-beads (FIG. 30). In all of these cases, the hybridization captureoligonucleotide HCO, or the covalent ligation capture oligo constructLCO, is covalently attached to the inner surface of reaction tubes andplates, spotted on the glass (plastic) surface to create a micro-array,or cover the surface of micro-beads.

The synthesized and surface-immobilized Whole Genome Library (asdescribed above), or Whole Methylome Library (if themethylation-sensitive restriction nucleases, such as Aci I, Acc II, AspLE I, Ava I, Bce AI, Bsa HI, Bsh 1236 I, Bsi E1, Bsi SI, Bst FN I, BstHH I, Bst UI, Cfo I, Hap II, Hga I, Hha I, HinP1 I, Hin 6I, Hpa II, Hpy99I, Hpy CH4 IV, Hsp AI, Mvn I, and Ssi I, or the methylation-specificnucleases, such as McrBC, are included into the Enx-O-Mix or used afterthe library synthesis) can be further processed enzymatically, washed,and amplified or stored in an immobilized format. FIG. 31 illustrates aspecific but exemplary use of the one-step library synthesis andimmobilization in a hypothetical fluidic device. The process starts by alibrary synthesis (I), is continued by a library immobilization at thebottom of a reaction vessel (II), and is completed by removal of thefirst reagent mix, washing, and introduction of a new reagent mix (forexample, PCR components).

Example 8 Enz-O-Mix DNA Library Preparation and SimultaneousAmplification by Strand Displacement Synthesis

This example demonstrates an exemplary preparation of a library fromcell-free urine DNA by ligation of hairpin oligonucleotide adaptorcomprising deoxy-uridine and N.BbvC IB nicking endonuclease recognitionsite located at the loop region as illustrated in FIG. 16A followed byisothermal amplification with a strand-displacing DNA polymerase (seeFIG. 6A and FIG. 19A, Enz-O-Mix 3). The adaptor oligonucleotide used inthis example (Table I, SEQ ID NO:18) is based on the adaptor sequencedescribed in Example 3 (Table I, SEQ ID NO:8), except that it hasrecognition sequence 5′-CCTCAGC-3′ for N.BbvC IB nicking endonuclease inthe loop region. In the first step, a stem-loop adaptor attachment isaccomplished by using a mix (FIG. 18, Master Mix III) with 3 enzymaticactivities: T4 DNA ligase, T4 DNA polymerase, and Uracil-DNAglycosylase. UDG creates abasic sites by destabilizing the 5′ end of theadaptor stem. T4 DNA polymerase generates blunt ends at the restrictionfragments leaving intact 5′ phosphate groups that are necessary forligation to the free 3′ end of the adaptor by the T4 DNA ligase. T4 DNApolymerase then extends the 3′ ends of the newly ligated restrictionfragments into the adaptor, thereby displacing the 5′ end of theadaptor's stem until an abasic site generated by UDG is reached thatstops further extension. By doing so, the T4 DNA polymerase generates afunctional double-stranded N.BbvC IB nicking endonuclease recognitionsite. In the second step, following thermal inactivation of T4 DNApolymerase and T4 DNA ligas, the mix is supplemented with two additionalenzymatic activities, N.BbvC IB nicking endonuclease generatingsingle-stranded nicks at the adaptor (and internal) sites, and thestrand-displacing Klenow fragment of DNA polymerase I, initiating stranddisplacement from the nicks. The newly synthesized strand is extendeduntil a nick or abasic site at the template strand is encountered. Inorder to fill-in a second strand and to recreate intact N.BbvC IBrecognition sites, an oligonucleotide primer comprising the adaptor'sloop sequence at its 5′-end plus five bases complementary to theadaptor's stem at its 3′-end (Table I, SEQ ID NO:19) is also added tothe mix. As a result, a self-sustained isothermal DNA amplificationprocess is accomplished.

Cell-free DNA was isolated from urine of healthy donors as described inExample 5. Aliquots of 50 nanograms of DNA were incubated in 1× NEBuffer4 (New England Biolabs; Beverly, Mass.) with 2 μM adaptoroligonucleotide comprising deoxy-uridine and N.BbvC IB nickingendonuclease recognition site (Table I, SEQ ID NO:18); 1 mM ATP; 40 μMeach dNTP; 200 μg/ml BSA; 1 unit of uracil-DNA glycosylase (New EnglandBiolabs; Beverly, Mass.); 0.18 unit of T4 DNA polymerase (New EnglandBiolabs; Beverly, Mass.); and 600 units of T4 DNA ligase (New EnglandBiolabs; Beverly, Mass.) for 1 hour at 37° C. in a final volume of 15μl. A negative control containing no T4 DNA ligase was run in parallel.Samples were heated at 65° C. for 15 mM, cooled to 37° C. andsupplemented with 10 units of N.BbvC IB nicking endonuclease (NewEngland Biolabs; Beverly, Mass.); 27 ng/μl of E. coli Single StrandedBinding Protein (SSB protein, USB Corporation, Cleveland, Ohio); 5 unitsof Klenow Exo-fragment of DNA polymerase I (USB Corporation, Cleveland,Ohio); 0.5 μM of dU_N.BbvC IB Nick Primer (Table I, SEQ ID NO:19); and200 μM of each dNTP in a final volume of 30 μl of 1× NEBuffer 4 (NewEngland Biolabs; Beverly, Mass.). To test the effect of additives on therepresentation of GC-rich sequences such as promoter sites, reactionscontaining 4% of dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis,Mo.) and 7deaza-dGTP (Roche Diagnostics, Indianapolis, Ind.) were run inparallel. A control reaction containing no N.BbvC IB nickingendonuclease was also included. Samples were incubated for 1 to 3 hoursat 37° C. and reactions were stopped by adding 50 mM of EDTA. Productsof the library amplification were analyzed by gel electrophoresis on 20%acrylamide TBE gel (Invitrogen Corporation, Carlsbad, Calif.), afterstaining with Sybr Gold (Molecular Probes; Carlsbad, Calif.).

FIG. 29 shows the products of an exemplary isothermal DNA amplificationafter 1 hour incubation. As shown, in the presence of all five enzymaticactivities the lambda restriction fragments are converted into a libraryamplified as DNA products, whereas in the absence of T4 DNA ligase orN.BbvC IB nicking endonuclease no accumulation of DNA occurs.

Example 9 Integrated Whole Genome Amplification in a Closed-TubeReaction Using Degradable Hairpin Adaptor Comprising Deoxy-Uridine

Traditionally, preparation of DNA libraries and their subsequentamplification by PCR involves multiple separate processes and differentbuffer systems. Previous embodiments and examples of this invention havedemonstrated that Enz-O-Mix approach can substantially simplify theseprocesses and reduce number of steps necessary for WGA and WMA librarypreparation to one step. However, in previous Examples the Enz-O-Mix DNAlibraries were first, prepared in small volume (15μl) of buffer A (NEB4), and then amplified by PCR in a larger volume (75 ml) of buffer B(Titanium Taq reaction buffer), FIG. 30A. In all of these cases,Enz-O-Mix library synthesis/amplification is performed in twooperational steps: step 1—a tube containing DNA is supplemented with thelibrary (WGA or WMA) synthesis reagents and incubated at 37° C. for 1 h,and step 2—a tube is opened, supplemented with PCR amplificationbuffer/reagents and subjected to temperature cycling. Such an approachis referred to herein as a two-step, opened-tube protocol.

This example introduces further simplification in preparation andPCR-mediated amplification of WGA/WMA libraries when all the necessarysynthesis/amplification reagents are introduced into the tube priorreaction, and library synthesis and subsequent amplification occur inthe same volume and the same buffer without opening the tube within apre-programmed thermocycler (FIG. 30B). Such an approach is referred toherein as an integrated, one-step, closed-tube protocol. Due to itssimplicity and lack of any human intervention, the integrated, one-step,closed-tube Enz-O-Mix DNA amplification can be easily automated and usedfor high-throughput research applications and clinical diagnostics.

FIG. 31 shows a one possible temperature profile (b) and an envisionedDNA accumulation during amplification (a), where incubation at 37° C.for 1 h (DNA library synthesis) is followed by heating at 95° C. for 10min (Taq DNA polymerase activation and stem-loop adaptor→universal PCRprimer conversion), and then by thermocycling between 65° C. and 95° C.for 1 h (PCR-mediated whole genome or whole methylome amplification).

FIG. 32 shows biochemical and physicochemical reactions involved in thetransformation of the stem-loop adaptor oligonucleotide into afunctional universal PCR primer adequate for efficient amplification ofsynthesized WGA/WMA libraries. Dual utilization of the stem-loopoligonucleotide as adaptor and PCR primer eliminate a necessity tointroduce into reaction an additional single stranded oligo-primer (dueto the 3′ profreading activity of a DNA polymerase involved in thelibrary synthesis process such primer should contain at least severalnuclease-resistant bases at its 3′ terminus).

In this example, a WGA process is described wherein a hairpinoligonucleotide adaptor comprising deoxy-uridine described in Example 3is ligated via its free 3′ end to the 5′ phosphates of DNA restrictionfragments generated from intact genomic DNA in a single enclosedcontainer exemplary reaction mix comprising 6 exemplary enzymaticactivities: AluI restriction endonuclease, RsaI restrictionendonuclease, T4 DNA ligase, T4 DNA polymerase, a hot-start Taq DNApolymerase, and Uracil-DNA glycosylase (UDG). In an initial isothermalincubation UDG catalyses the release of free uracil and generates abasicsites in the adaptor's loop region and the 5′ half of the hairpin. AluIand RsaI digest the target DNA into restriction fragments. T4 DNApolymerase generates blunt ends of the restriction fragments. T4 DNAligase generates a phosphodiester bond between the 3′ ends of theadaptor molecules and the 5′ phosphates of the restriction fragments. T4DNA polymerase extends the free 3′ end of the restriction fragmentsusing as template the ligated 3′ end of the hairpin stem until an abasicsite is reached that serves as a replication stop (FIG. 10B). Samplesare heated to 72° C. to inactivate all thermo-labile enzymes and toactivate Taq polymerase, then to 95° C. to degrade the abasic sites ofthe adaptor and to generate active primer from the remaining intactstrand of the adaptor that is free of abasic sites. The resultinglibrary is finally amplified by thermal cycling. The entire processtakes place in a single enclosed reaction container under programmedtemperature control algorithm and without any intermediate liquidhandling.

Ten nanogram aliquots of human genomic DNA isolated from the peripheralblood of a healthy donor by standard procedures were incubated in anexemplary reaction mix comprising: 1× Titanium Taq buffer (Clontech,Mountain View, Calif.); 0.6 μl of Titanium Taq (Clontech, Mountain View,Calif.); 2 μM dU Hairpin Adaptor oligonucleotide (Table I, SEQ ID NO:7);1 mM ATP; 200 μM dNTPs; 0.36 Units of T4 DNA polymerase; 1200 units ofT4 DNA ligase; 2 Units of UDG; 10 units each of AluI and RsaIrestriction endonucleases (New England Biolabs; Beverly, Mass.), and1:10,000 dilutions of fluorescein and SybrGreen I (Molecular Probes;Carlsbad, Calif.) in a final volume of 30 μA To study the effect of Mg⁺⁺ions and DMSO, reactions supplemented with MgCl₂ at final concentrationsof 5 mM and 7.5 mM and with DMSO at a final concentration of 4% werealso included (It should be noted that the 1× Titanium Taq buffercontains 3.5 mM MgCl₂). Reactions were incubated at 37° C. for 1 hr,followed by 72° C. for 10 min, 95° C. for 10 min, and 11 cycles of 94°C. for 20 sec and 65° C. for 2 min. on i-Cycler real-time PCR instrument(Bio-Rad; Hercules, Calif.). FIG. 33 shows the amplification curves ofthe resulting libraries. As shown, Mg⁺⁺ concentrations of 5 mM and 7.5mM in the reaction buffer supported the enzymatic activities present inthe mix better than the basic Titanium Taq buffer containing 3.5 mMMgCl₂. The presence of 4% DMSO did not have significant effect on theWGA amplification when higher Mg⁺⁺ concentrations were applied.

Example 10 Hot Start PCR Using Degradable Stem-Loop Primers

Specificity and the ability to amplify a single DNA or RNA target is oneof the most important requirements for application of PCR in moleculardiagnostics. Hot start PCR protocol was introduced to reduce thenon-specific primer/template and primer/primer annealing events thatoccur at lower temperatures and subsequently result in non-specificamplification products. Several methods and corresponding commercialproducts for performing hot start PCR rely upon the physical separationof PCR reagents until the high temperature of the reaction has beenreached. Those products include the following:

1. Wax beads (Ampliwax PCR Gems, Perkin Elmer) that create a temporarybarrier between dNTPs, buffer, and MgCl₂ on one side of the wax layer,and DNA template and DNA polymerase on another side.

2. Small beads of wax with encapsulated Taq DNA polymerse (Taq Bead HotStart Polymerase, Promega). In this case Taq DNA polymerase is releasedwhen the reaction reaches 60° C.

3. Small beads of wax with encapsulated magnesium (StartaSpere,Stratagene).

4. Taq DNA polymerase inactivated by antibody (JumStart Taq DNApolymerase, Sigma; TaqStart and TthStart, Clontech; AmpliTaq Gold, PEBiosystems; HotStarTaq, Qiage; etc.). Antibody binds to the polymeraseand inactivates it at low temperature but denatures and releases theactive polymerase at high temperature.

Example 9 introduced the idea of using a degradable, dU-containingstem-loop oligonucleotide-adaptor as a universal PCR primer (see FIG.32) for whole genome amplification. This embodiment extends the use ofdegradable, dU-containing stem-loop oligonucleotides as “hot start” PCRprimers for locus-specific DNA amplification.

As shown in FIG. 38 and FIG. 39, both PCR primers are synthesized in aform of stem-loop oligonucleotides A and B. The 3′ portion of thestem-loop oligonucleotides represents the primer sequence (no dU bases);the 5′ portion represents the sequence complementary to the primersequence and comprises one or more dU bases substituting dT bases; andthe loop comprises several dT and dU bases.

PCR reaction is assembled by mixing together DNA, dNTPs, magnesium, PCRbuffer, Taq DNA polymerase (or any other thermostable DNA polymerase),and dU-glycosylase (FIG. 38 and FIG. 40). A thermocycler is programmedto have the following conditions:

a) Incubation step at 37° C. for 15-30 min that is necessary to convertall dU bases within the stem-loop oligonucleotides A and B into theabasic sites;

b) Incubation step at 95° C. for 10 min that is necessary to denatureDNA, introduce breaks at the abasic sites within the stem-loopoligonucleotides A and B, and thus release the active primers A and B;

c) A regular PCR cycling mode that amplifies the DNA region defined bythe primer A and B.

The conversion of the stem-loop oligonucleotides A and B into PCRprimers A and B (see FIG. 39) results from several breaks introduced byheating the loop and the 5′ portion of the oligonucleotides. At hightemperature, the small oligonucleotides originated from the 5′ stemregions can not form stable interaction with the remaining intact 3′primer regions and do not affect the PCR process. In general, theposition of dU bases within the 5′ stem region is dictated by thelocation of thymines in a DNA sequence, although they can be alsointroduced at another nucleotide position (thus generating a stem withone ore several mismatched bases). The number of dU bases within the 5′stem and the loop regions can vary from 1 to about 6, and an optimalnumber can be determined empirically.

The proposed hot start primer method can be used in PCR and otherDNA/RNA amplification methods that utilize thermostable polymerases. Ithas several advantages over the hot start PCR methods that involveeither non-degradable hairpin primers with short stem [Kaboev, O. K., etal., 2000; Ailenberg, M., and Silverman, M., 2000], or duplex primers[Kong, D., et al., 2004]: a) the hybridization kinetics are notcompromised by the presence of a stem region (as it might be in the caseof non-degradable stem-loop primer method); b) no inverted repeat isformed at the ends of PCR amplicons that could substantially reduce theefficiency of the PCR amplification process (as it happens in the caseof non-degradable stem-loop primer method); and c) oligonucleotidescomplementary to the priming oligonucleotide (products of the stem-loopoligonucleotide fragmentation) are short, do not form stable interactionwith the remaining intact 3′ primer regions and, as a result, do notaffect the PCR process (as it possible in the duplex primer method).Advantages over other existing hot-start methods include but are notlimited to the following: a) no phase separation is necessary and allreaction components are originally present in the same reaction mixsimplifying storage and reducing production cost; b) no expensiveblocking antibodies are involved; c) no mutagenesis or chemicalmodification of amino acid residues in the thermostable DNA polymeraseare required whatsoever.

REFERENCES

All patents and publications mentioned in the specification areindicative of the level of those skilled in the art to which theinvention pertains. All patents and publications are herein incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by reference.

PATENTS AND PATENT APPLICATIONS

U.S. Pat. No. 6,777,187

U.S. patent application Ser. No. 10/797,333

U.S. patent application Ser. No. 10/795,667

U.S. patent application Ser. No. 11/071,864

PUBLICATIONS

Barns, W. M. PCR amplification of up to 35-kb DNA with high fidelity andhigh yield from lambda bacteriophage templates. Proc. Natl. Acad. Sci.USA, 91, 2216-2220, (1994).

Broude, N.E., Stem-loop oligonucleotides: a robust tool for molecularbiology and biotechnology. Trends in Biotechnology, 20, 249-256, (2002).

de Baar, M. P., et al. One-tube real-time isothermal amplification assayto identify and distinguish human immunodeficiency using type I subtypesA, B, and C and circulating recombinant forms AE and AG. J. Clin.Microbiol. 39, 1895-1902, (2001).

Deiman, B., et al. Characteristics and applications of nucleic acidsequence-based amplification (NASBA). Mol. Biotechnology, 20, 163-179,(2002); Hill, C. S., Molecular diagnostic testing for infectiousdiseases using TMA technology. Expert Rev Mol Diagn, 1, 445-455, (2001).

Goddard, N. L., et al. Sequence dependent rigidity of single strandedDNA. Phys. Rev. Lett. 85, 2400-2403, (2000).

Hamad-Schifferli, K., et al. Remote electronic control of DNAhybridization through inductive coupling to an attached metalnanocrystal antenna. Nature, 415, 152-155, (2002).

Hellyer, T. J., and Naolean, J. G., Strand displacement amplification: aversatile tool for molecular diagnostics. Expert Rev Mol Diagn, 4,251-261, (2004).

Kaboev, O. K., et al. PCR hot start using primers with the structure ofmolecular beacons (stem-loop-like structure). Nucleic Acids Res., 28,e94, (2000); Ailenberg, M., and Silverman, M. Controlled hot start andimproved specificity in carrying out PCR utilizing touch-up and loopincorporated primers (TULIPS). Bio-Techniques, 29, 1018-1024, (2000).

Kong, D., et al. PCR hot-start using duplex primers. Biotechnol. Lett.26, 277-280, (2004).

Liu, et al. Molecular beacons for DNA biosensors with micrometer tosubmicrometer dimensions. Anal. Biochem. 283, 56-63, (2000).

Mackay, I. M., et al. Real-time PCR in virology. Nucleic Acids Res., 30,1292-1305, (2002); Elnifro, E. M., et al. Multiplex PCR: optimizationand application in diagnostic virology. Clin. Microbiol. Rev. 13,559-570, (2000).

Riccelli, P. V., et al. Hybridization of single-stranded DNA targets toimmobilized complementary DNA probes: comparison of stem-loop versuslinear capture probes. Nucleic Acids Res., 29, 996-1004, (2001).

Sokol, D. L., et al. Real-time detection of DNA-RNA hybridization inliving cells. Proc. Natl. Acad. Sci. USA, 95, 11538-11543, (1998).

Summerer, D., and Marx, A., A molecular beacon for quantitativemonitoring of the DNA polymerase reaction in real-time. Angew. Chem.Int. 41, 3620-3622, (2002).

Tyagi, S., and Kramer, F. R., Molecular beacons: probes that fluoresceupon hybridization. Nat Biotechnol. 14, 303-308, (1996).

Tyagi, S., et al. Multicolor molecular beacons for allelediscrimination. Nat. Biotechnol. 16, 49-53, (1998).

van Deursen, P. B. H., et al., A novel quantitative multiplex NASBAmethod: application to measuring tissue factor and CD14 mRNA levels inhuman monocytes. Nucleic Acids Res, 27, e15, (1999).

Whitcombe, D., et al. Detection of PCR products using self-probingamplicons and fluorescence. Nat. Biotechnol. 17, 804-807, (1999)

Although the present invention and its advantages have been described indetail, it should be understood that various changes, substitutions andalterations can be made herein without departing from the spirit andscope of the invention as defined by the appended claims. Moreover, thescope of the present application is not intended to be limited to theparticular embodiments of the process, machine, manufacture, compositionof matter, means, methods and steps described in the specification. Asone of ordinary skill in the art will readily appreciate from thedisclosure of the present invention, processes, machines, manufacture,compositions of matter, means, methods, or steps, presently existing orlater to be developed that perform substantially the same function orachieve substantially the same result as the corresponding embodimentsdescribed herein may be utilized according to the present invention.Accordingly, the appended claims are intended to include within theirscope such processes, machines, manufacture, compositions of matter,means, methods, or steps.

What is claimed is:
 1. A method of preparing a nucleic acid molecule,comprising: providing a double stranded nucleic acid DNA molecule; andattaching one strand of a stem-loop oligonucleotide comprising aninverted repeat and a loop to the double stranded nucleic acid moleculeto produce an oligonucleotide-attached nucleic acid molecule, whereinthe stem-loop oligonucleotide comprises at least one deoxy-uridine. 2.The method of claim 1, wherein the attaching is further defined asattaching the oligonucleotide to the double stranded nucleic acidmolecule under conditions to produce a non-covalent junction in theoligonucleotide-attached nucleic acid molecule.
 3. The method of claim2, wherein the non-covalent junction comprises a nick, a gap, or a 5′flap structure.
 4. The method of claim 1, wherein the attaching isfurther defined as ligating.
 5. The method of claim 1, furthercomprising displacing one strand of the oligonucleotide from theoligonucleotide-attached nucleic acid molecule by strand displacement orby nick translation polymerization.
 6. The method of claim 1, furthercomprising amplifying at least part of the oligonucleotide-attachednucleic acid molecule.
 7. The method of claim 6, wherein the at leastpart of the oligonucleotide-attached nucleic acid molecule is amplifiedby polymerase chain reaction.
 8. The method of claim 6, wherein the atleast part of the oligonucleotide-attached nucleic acid molecule isamplified by RNA transcription.
 9. The method of claim 6, wherein the atleast part of the oligonucleotide-attached nucleic acid molecule isamplified by strand displacement.
 10. The method of claim 1, furthercomprising amplifying the oligonucleotide-attached nucleic acidmolecule, wherein at least part of the inverted repeat is excluded fromthe amplified oligonucleotide-attached nucleic acid molecule.
 11. Themethod of claim 4, wherein the ligating is further defined ascomprising: generating ligatable ends on the double stranded nucleicacid molecule; generating a ligatable end on the stem-loopoligonucleotide; and ligating one strand of the ligatable end of thestem-loop oligonucleotide to one strand of an end of the nucleic acidmolecule, thereby generating a non-covalent junction in theoligonucleotide-attached nucleic acid molecule.
 12. The method of claim1, further defined as comprising: generating blunt ends on the nucleicacid molecule; generating a blunt end on the stem-loop oligonucleotide;and ligating one strand of the blunt end of the stem-loopoligonucleotide to one strand of a blunt end of the nucleic acidmolecule, thereby generating a nick in the oligonucleotide-ligatednucleic acid molecule.
 13. The method of claim 1, wherein said stem-loopoligonucleotide comprises a defined sequence.
 14. The method of claim 1,wherein said stem-loop oligonucleotide comprises a transcriptionregulatory sequence.
 15. The method of claim 1, further comprising:digesting the DNA molecule with one or more endonucleases to produce DNAfragments; producing blunt ends on the DNA fragments; producing a bluntend on the stem-loop oligonucleotide; and ligating one strand of theblunt end of a stem-loop oligonucleotide to one strand of a blunt end ofa DNA fragment, thereby generating a nick in a oligonucleotide-ligatedDNA fragment.
 16. The method of claim 15, wherein the endonuclease is arestriction endonuclease, DNAse I, or an apoptotic endonuclease.
 17. Themethod of claim 16, wherein the restriction endonuclease ismethylation-specific or methylation-sensitive.
 18. The method of claim1, wherein the oligonucleotide-attached nucleic acid molecule comprisesa nick having a 3′ hydroxy group.
 19. The method of claim 5, wherein thestrand displacement or nick translation polymerization is furtherdefined as polymerization that ceases at a non-replicable base or regionin the loop or in a region of the stem adjacent to the loop.
 20. Themethod of claim 1, further comprising the step of digesting the doublestranded DNA molecule with an endonuclease to generate DNA fragments.21. The method of claim 1, wherein the stem-loop oligonucleotide isfurther defined as comprising a further non-replicable base or sequencein addition to the at least one deoxy-uridine.
 22. The method of claim21, wherein at least part of the non-replicable base or sequence ispresent in the loop of the oligonucleotide or in a sequence of the stemadjacent to the loop.
 23. The method of claim 21, wherein thenon-replicable base or sequence comprises an abasic site or sequence,hexaethylene glycol, or a bulky chemical moiety attached to thesugar-phosphate backbone or the base.
 24. The method of claim 23,wherein the non-replicable base or sequence comprises an abasic site orsequence that is introduced by one or more enzymes.
 25. The method ofclaim 1, wherein the loop of the stem-loop oligonucleotide comprises atleast one deoxy-uridine.
 26. The method of claim 5, wherein the stranddisplacement or nick translation polymerization of theoligonucleotide-attached nucleic acid molecule generates an endonucleasesite.
 27. The method of claim 26, wherein the endonuclease site is asite-specific restriction endonuclease site and wherein at least part ofthe inverted repeat is removed by cleavage with said restrictionendonuclease.
 28. The method of claim 26, wherein the endonuclease siteresides within the stem and loop regions.
 29. The method of claim 26,wherein the endonuclease site resides within the loop region.
 30. Themethod of claim 26, wherein the endonuclease site resides within thestem region.
 31. The method of claim 26, wherein the endonuclease siteis a homing endonuclease site.
 32. The method of claim 14, furthercomprising initiating RNA transcription from the transcriptionregulatory sequence, thereby producing at least one transcribedpolynucleotide.
 33. The method of claim 32, wherein the transcribedpolynucleotide is replicated by a reverse transcriptase.
 34. The methodof claim 33, wherein reverse transcription is initiated by hybridizationof an oligonucleotide complementary to the 3′ end of the transcribedpolynucleotide.
 35. The method of claim 1, wherein the stem-loopoligonucleotide further comprises a recognition sequence for a nickingendonuclease and wherein the oligonucleotide-attached nucleic acidmolecule is amplified by strand displacement synthesis and second strandsynthesis.
 36. The method of claim 35, wherein the recognition sequencefor a nicking endonuclease resides at least in part within the stem ofthe stem-loop oligonucleotide.
 37. The method of claim 35, wherein therecognition sequence for a nicking endonuclease resides at least in partwithin the loop of the stem-loop oligonucleotide.
 38. The method ofclaim 1, wherein a 5′ end of the stem-loop oligonucleotide lacks aphosphate.
 39. The method of claim 1, further comprising incorporatingthe nucleic acid molecule into a vector.
 40. The method of claim 39,wherein the nucleic acid is incorporated at ends in the modifiedmolecule generated by endonuclease cleavage sites within the invertedrepeat.
 41. The method of claim 1, wherein the method is carried out ina single suitable solution.
 42. The method of claim 41, wherein themethod is carried out at one temperature.
 43. The method of claim 41,wherein the solution comprises one or more of the following: ligase; DNApolymerase; one or more endonucleases; RNA polymerase; reversetranscriptase; RNase H; deoxy-uridine glycosylase; nickase; thermophilicDNA polymerase; ATP; rNTPs; and dNTPs.
 44. The method of claim 6,wherein the method is carried out in a single suitable solution.
 45. Themethod of claim 1, wherein the oligonucleotide-attached nucleic acidmolecule is immobilized to a solid support.
 46. The method of claim 45,wherein the molecule is immobilized non-covalently.
 47. The method ofclaim 45, wherein the molecule is immobilized covalently.
 48. The methodof claim 1, wherein the stem-loop oligonucleotide comprises more thanone deoxy-uridines.